Mouse CD40 ligand-specific antibodies and hybridomas

ABSTRACT

There is disclosed a polypeptide (CD40-L) and DNA sequences, vectors and transformed host cells useful in providing CD40-L polypeptides. More particularly, this invention provides isolated human and murine CD40-L polypeptides that bind to the extracellular binding region of a CD40 receptor.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of application Ser. No. 08/249,189,filed May 24, 1994, now U.S. Pat. No. 5,961,974; which in turn is aContinuation-In-Part of application Ser. No. 07/969,703, filed Oct. 23,1992 (now abandoned); which in turn is a Continuation-In-Part ofapplication Ser. No. 07/805,723, filed Dec. 5, 1991 (now abandoned);which in turn is a Continuation-In-Part of application Ser. No.07/783,707, filed Oct. 25, 1991 (now abandoned).

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel cytokine. More specifically,the present invention relates to the cloning of a murine and a humancytokine that binds to a human CD40 having both agonist and antagonistactivity in soluble and membrane-bound forms.

BACKGROUND OF THE INVENTION

Cytokines that have an “Interleukin” designation are those proteinfactors that influence immune effector cells. Cytokines designatedinterleukin-1 through interleukin-12 have been reported and named as aninterleukin. Other known cytokines include tumor necrosis factor (TNF),granulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), mast cell growth factor (MGF),epidermal growth factor (EGF), platelet-derived growth factor (PDGF),nerve growth factor (NGF), erythropoietin (EPO), γ-interferon (γ-IFN)and others.

DNAs for two different TNF receptors (Type I and Type II) have beencloned (Smith et al., Science 248:1019, 1990; and Schall et al., Cell61:361, 1990). Both forms of TNF receptor are related to each other andbelong to a family of receptors whose members include nerve growthfactor receptor (Johnson et al., Cell 47:545, 1986), B cell antigen CD40(Stamenkovic et al., EMBO J. 8:1403, 1989), T cell antigen OX40 (Mallettet al., EMBO J. 9:1063, 1990), human Fas antigen (Itoh et al., Cell66:233, 1991) and murine 4-1BB receptor (Kwon et al., Cell. Immunol.121:414, 1989 [Kwon et al. I] and Kwon et al., Proc. Natl. Acad. Sci.USA 86:1963, 1989 [Kwon et al. II]).

Human CD40 protein (CD40) is a peptide of 277 amino acids having amolecular weight of 30,600, and a 19 amino acid secretory signal peptidecomprising predominantly hydrophobic amino acids (Stamenkovic et al.).The molecular weight (exclusive of glycosylation) of the mature humanCD40 protein is 28,300. A cDNA encoding human CD40 was isolated from acDNA library prepared from Burkitt lymphoma cell line Raji. The putativeprotein encoded by the CD40 cDNA contains a putative leader sequence,trans-membrane domain and a number of other features common tomembrane-bound receptor proteins. CD40 has been found to be expressed onB lymphocytes, epithelial cells and some carcinoma cell lines.

A monoclonal antibody (mAb) directed against CD40 has been shown tomediate various functional effects of human B cells. These effectsinclude: (a) homotypic adhesions (Gordon et al., J. Immunol. 140:1425,1988 [Gordon et al. I]); (b) increased cell size (Gordon et al. I andValle et al., Eur. J. Immunol. 19:1463, 1989); (c) proliferation of Bcells activated with anti-IgM, anti-CD20 mAb, phorbol ester alone (Clarket al., Proc. Natl. Acad. Sci. USA 83:4494, 1986; and Paulie et al., J.Immunol. 142:590, 1989), or phorbol ester combined with interluekin-4(Gordon et al., Eur. J. Immunol. 17:1535, 1987 [Gordon et al. II]; and(d) production of IgE (Jabara et al., J. Exp. Med. 172:1861, 1990; Zhanget al., J. Immunol. 146:1836, 1991) and IgM (Gascan et al., J. Immunol.147:8, 1991) from interluekin-4 (IL-4) stimulated T-depleted cultures.

One such antibody, called mAb 89 by Banchereau et al., Clin. Immunol.Spectrum 3:8, 1991 [Banchereau et al. I], was found to induce human Bcell proliferation at a relatively low antibody concentration (30 ng/mlor about 10⁻¹⁰ M). Proliferation lasted two to three weeks and resultedin a ten-fold expansion of the human B cell population. Optimalstimulation of the B cells occurred when CD40 surface molecule wascross-linked by IgM. Fab fragments of another anti-CD40 mAb induced onlya weak proliferative response. Further, Banchereau et al., Science251:70, 1991 [Banchereau et al. II] reported that resting human B cellsentered a state of sustained proliferation when incubated with both amurine fibroblastic Ltk⁻ cell line that was transfected with human Fcreceptor and with a monoclonal antibody specific for human CD40.Banchereau et al. II found that cross-linking CD40 is necessary forclonal expansion of B cells.

CD23 is a low affinity IgE receptor that has been found to be expressedon most IgM⁻/IgD⁻ mature B cells, but not T cells. CD23 has beensequenced and its sequence was described in Kikutani et al., Cell47:657, 1986. Soluble CD23 (sCD23) was found to induce a pyrogenicreaction in rabbits and this reaction was abrogated by administration ofhuman IgE (Ghaderi et al., Immunology 73:510, 1991). Therefore, CD23 maybe an appropriate marker for soluble CD40 or CD40-L effects.

Prior to the present invention, a ligand for CD40 was unknown.Accordingly, there is a need in the art to identify and characterize aCD40 ligand (CD40-L).

SUMMARY OF THE INVENTION

A novel cytokine, hereafter referred to as “CD40-L,” has been isolatedand characterized. The nucleotide sequence and deduced amino acidsequence of representative murine CD40-L cDNA is disclosed in SEQ IDNO:1 and FIG. 1, and the amino acid sequence is also listed in SEQ IDNO:2. The nucleotide sequence and deduced amino acid sequence ofrepresentative human CD40-L cDNA is disclosed in SEQ ID NO:11 and FIG.2, and the amino acid sequence is also listed in SEQ ID NO:12. Thepresent invention further comprises other CD40-L polypeptides encoded bynucleotide sequences that hybridize, under moderate or severe stringencyconditions, to probes defined by SEQ ID NO:11 (the coding region ofhuman CD40-L), fragments of the sequence extending from nucleotide 46 tonucleotide 828 of SEQ ID NO:11, or to DNA or RNA sequences complementaryto FIG. 2 (SEQ ID NO:11) or fragments thereof. The invention furthercomprises nucleic acid sequences which, due to the degeneracy of thegenetic code, encode polypeptides substantially identical orsubstantially similar to polypeptides encoded by the nucleic acidsequences described above, and sequences complementary to them.

CD40-L is a type II membrane polypeptide having an extracellular regionat its C-terminus, a transmembrane region and an intracellular region atits N-terminus. A soluble version of murine CD40-L has been found insupernatants from EL-4 cells and EL-4 cells sorted on the basis of abiotinylated CD40/Fc fusion protein described herein. Soluble CD40-Lcomprises an extracellular region of CD40-L or a fragment thereof. Theprotein sequence of murine CD40-L is described in FIG. 1 and SEQ IDNO:2, and human CD40-L in FIG. 2 and SEQ ID NO:12. The extracellularregion of murine CD40-L extends from amino acid 47 to amino acid 260 inFIG. 1 and SEQ ID NO:2, and of human CD40-L from amino acid 47 to aminoacid 261 in FIG. 2 and SEQ ID NO:12. CD40-L biological activity ismediated by binding of this cytokine with CD40 and includes B cellproliferation and induction of antibody secretion, including IgEsecretion.

The present invention further provides antisense or senseoligonucleotides (deoxyribonucleotides or ribonucleotides) thatcorrespond to a sequence of at least about 12 nucleotides selected fromthe nucleotide sequence of CD40-L or DNA or RNA sequences complementaryto the nucleotide sequence of CD40-L as described in SEQ ID NO:1 and SEQID NO:11 and in FIGS. 1 and 2. Such antisense or sense oligonucleotidesprevent transcription or translation of CD40-L mRNA or polypeptides.

Further still, the present invention provides CD40-L peptide fragmentsthat correspond to a protein sequence of at least 10 amino acidsselected from the amino acid sequence encoded by SEQ ID NO:1 or SEQ IDNO:11 that can act as immunogens to generate antibodies specific to theCD40-L immunogens. Such CD40-L immunogen fragments can serve asantigenic determinants in providing monoclonal antibodies specific forCD40-L.

The invention also provides a human CD40/Fc fusion protein and a solubleCD40 protein (sCD40) comprising the extracellular region of human CD40.Both sCD40 and CD40/Fc fusion protein can inhibit CD40-L or anti-CD40mAb induced B cell stimulation, IL-4-induced IgE stimulation and IL-4induced CD23 induction in B cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) illustrates nucleotide and aminoacid sequences corresponding to murine CD40-L. This protein is a type IIpolypeptide having its N-terminus as its intracellular domain, followedby a transmembrane region, and an extracellular domain at the C-terminusof the polypeptide. The extracellular domain, which is longer thaneither the intracellular domain or the transmembrane region, containsone potential N-linked glycosylation site and two potential disulfidebonds in view of four cysteine (Cys) residues.

FIG. 2 (SEQ ID NO:11 and SEQ ID NO:12) illustrates nucleotide and aminoacid sequences corresponding to human CD40-L. This protein is a type IIpolypeptide having its N-terminus as its intracellular domain, followedby a transmembrane region, and an extracellular domain at the C-terminusof the polypeptide. The extracellular domain, which is longer thaneither the intracellular domain or the transmembrane region, contains 1potential N-linked glycosylation site and 2 potential disulfide bonds inview of 5 cysteine (Cys) residues.

FIG. 3 illustrates a comparison of protein sequences of human (SEQ IDNO:12) and murine (SEQ ID NO:2) CD40-L showing 77.7% homology at theamino acid level.

FIGS. 4A-4B illustrate proliferation of T cell depleted human peripheralblood mononuclear cells (PBMC) caused by incubation with CV1 cellstransfected with full length murine CD40-L cDNA (SEQ ID NO:1) andexpressing bound CD40-L (CD40-L⁺CV1 cells) when compared with CV1 cellstransfected with empty vector (HAVEO) and not expressing bound murineCD40-L. The day 7 proliferation results show that CD40-L⁺CV1 cellssignificantly increase proliferation of T-cell depleted PBMC in thepresence or absence of interleukin-4 (IL-4).

FIG. 5 illustrates a second determination of T cell depleted PBMCproliferation with addition of bound murine CD40-L and 10 ng/ml of IL-4.These data show no co-mitogenic effect of IL-4 but continued strongmitogenic effect of bound CD40-L.

FIG. 6 illustrates that bound CD40-L augments IgE secretion.

FIG. 7 illustrates that membrane-bound CD40-L stimulates CD23 sheddingin the presence of IL-4.

FIG. 8 illustrates proliferation of murine splenic B cells caused bymembrane-bound murine CD40-L or 7A1 cells, which is a helper T cellclone.

FIG. 9 illustrates a comparison of murine EL40.9 cells, a sorted cellline that was sorted on the basis of expression of murine CD40-L and Tcells 7A1 for induction of an antigen-specific response indicated byplaque forming cells (PFC) by anti-sheep red blood cells (SCBC).

FIG. 10 illustrates a comparison of B cell proliferative activity ofmembrane-bound CD40-L and other cell types transfected with differentcDNAs. Membrane-bound CD40-L showed significantly more B cellproliferative activity than a helper T cell clone or other controlcells.

FIG. 11 illustrates that 7C2 cells (a helper T cell clone) and CV1 cellstransfected with murine CD40-L cDNA induce anti SRBC plaque formingcells.

FIG. 12 illustrates a comparison of two helper T cell clones with cellsexpressing membrane-bound CD40-L for inducing murine B cellproliferation.

FIGS. 13A-13B illustrate induction of antigen-specific plaque formingcells by membrane-bound CD40-L and a helper T cell clone in the presenceor absence of added interleukin-2 (IL-2).

FIGS. 14A-14B show effects of membrane-bound CD40-L stimulating B cellproliferation and IgE secretion. The effects of membrane-bound CD40-Lwere inhibited by CD40 receptor but not by TNF receptor.

FIGS. 15A-15D show representative FACS profiles of peripheral blood Tcells stimulated for 16 hours with 10 ng/ml PMA and 500 ng/ml ionomycin,and stained with 5 μg/ml CD40/Fc, a control Fc protein, IL-4receptor/Fc, murine IgG₁, and a CD40-L monoclonal antibody referred toas M90.

FIGS. 16A-16D illustrate the ability of monoclonal antibodies M90 andM91 to inhibit binding of 2 μg/ml CD40/Fc to peripheral blood T cellsactivated as described for FIG. 15.

FIG. 17 demonstrates the ability of anti-CD40-L monoclonal antibodies tobind trimeric CD40-L and inhibit the ability of CD40-L to induce B cellproliferation. Purified tonsil B cells were cultured with 5 μg/mlimmobilized rabbit anti-human IgM and recombinant soluble human CD40-L.M90, M91 or an isotype control antibody were titrated into the cultures;incorporation of tritiated thymidine was used as a measure ofproliferation.

FIG. 18 presents the binding of trimeric human and murine CD40-L anddimeric human and murine CD40-L to C40/Fc as determined in a biosensorassay.

FIGS. 19A-19B illustrate the binding of trimeric human CD40-L and twopreparations of monomeric human CD40-L to C40/Fc as determined in abiosensor assay.

DETAILED DESCRIPTION OF THE INVENTION

Novel polypeptides that can act as a ligand for murine and human CD40have been isolated and sequenced. More particularly, cDNAs encodingthese ligands have been cloned and sequenced. Further provided aremethods for expression of recombinant CD40-L polypeptides. CD40-Lpolypeptide include other forms of mammalian CD40-L, such as derivativesor analogs of human or murine CD40-L. Murine and human CD40-L comprise a214 and 215, respectively amino acid extracellular region at theC-terminus of full length, membrane-bound polypeptide. The extracellularregion contains the domain that binds to CD40. Murine and human CD40-Lfurther comprise a homologous hydrophobic 24 amino acid transmembraneregion delineated by charged amino acids on either side and a 22 aminoacid intracellular region at their N-termini. The present inventionfurther comprises full length CD40-L polypeptides or fragments thereofcomprising all or part of the extracellular region or derivatives of theextracellular region and mammalian cells transfected with a cDNAencoding murine or human CD40-L and expressing human or murine CD40-L asa membrane-bound protein.

The present invention comprises isolated DNA sequences encoding CD40-Lpolypeptides and DNA or RNA sequences complementary to such isolated DNAsequences. The isolated DNA sequences and their complements are selectedfrom the group consisting of (a) nucleotides 184 through 828, 193through 828, 193 through 762, or 403 through 762 of the DNA sequence setforth in FIG. 2 (SEQ ID NO:11) and their complements, (b) DNA sequenceswhich hybridize to the DNA sequences of (a) or their complements underconditions of moderate stringency and which encode a CD40-L polypeptide,analogs or derivatives thereof, and (c) DNA sequences which, due to thedegeneracy of the genetic code, encode CD40-L polypeptides encoded byany of the foregoing DNA sequences and their complements. In addition,the present invention includes vectors comprising DNA sequences encodingCD40-L polypeptides and analogs, and host cells transfected with suchvectors.

The novel cytokine disclosed herein is a ligand for CD40, a receptorthat is a member of the TNF receptor super family. Therefore, CD40-L islikely to be responsible for transducing signal via CD40, which is knownto be expressed, for example, by B lymphocytes. Full-length CD40-L is amembrane-bound polypeptide with an extracellular region at its Cterminus, a transmembrane region, and an intracellular region at itsN-terminus. A soluble version of CD40-L can be made from theextracellular region or a fragment thereof and a soluble CD40-L has beenfound in culture supernatants from cells that express a membrane-boundversion of CD40-L. The protein sequence of the extracellular region ofmurine CD40-L extends from amino acid 47 to amino acid 260 in FIG. 1 andSEQ ID NO:2. The protein sequence of the extracellular region of humanCD40-L extends from amino acid 47 to amino acid 261 in FIG. 2 and SEQ IDNO:12. The biological activity of CD40-L is mediated by binding to CD40or a species-specific homolog thereof and comprises proliferation of Bcells and induction of immunoglobulin secretion from activated B cells.CD40-L (including soluble monomeric and oligomeric forms, as well asmembrane-bound forms) can effect B cell proliferation and immunoglobulinsecretion (except IgE secretion) without the presence of added IL-4, incontrast to anti-CD40 antibodies, which require IL-4 and cross-linkingto mediate activity.

CD40-L refers to a genus of polypeptides which are capable of bindingCD40, or mammalian homologs of CD40. As used herein, the term “CD40-L”includes soluble CD40-L polypeptides lacking transmembrane andintracellular regions, mammalian homologs of human CD40-L, analogs ofhuman or murine CD40-L or derivatives of human or murine CD40-L.

CD40-L may also be obtained by mutations of nucleotide sequences codingfor a CD40-L polypeptide. A CD40-L analog, as referred to herein, is apolypeptide substantially homologous to a sequence of human or murineCD40-L but which has an amino acid sequence different from nativesequence CD40-L (human or murine species) polypeptide because of one ora plurality of deletions, insertions or substitutions. Analogs of CD40-Lcan be synthesized from DNA constructs prepared by oligonucleotidesynthesis and ligation or by site-specific mutagenesis techniques.

The primary amino acid structure of human or murine CD40-L may bemodified to create CD40-L derivatives by forming covalent or aggregativeconjugates with other chemical moieties, such as glycosyl groups,lipids, phosphate, acetyl groups and the like, or by creating amino acidsequence mutants. Covalent derivatives of CD40-L are prepared by linkingparticular functional groups to CD40-L amino acid side chains or at theN-terminus or C-terminus of a CD40-L polypeptide or the extracellulardomain thereof. Other derivatives of CD40-L within the scope of thisinvention include covalent or aggregative conjugates of CD40-L or itsfragments with other proteins or polypeptides, such as by synthesis inrecombinant culture as N-terminal or C-terminal fusions. For example,the conjugate may comprise a signal or leader polypeptide sequence atthe N-terminal region or C-terminal region of a CD40-L polypeptide whichco-translationally or post-translationally directs transfer of theconjugate from its site of synthesis to a site inside or outside of thecell membrane or cell wall (e.g. the α-factor leader of Saccharomyces).CD40-L polypeptide fusions can comprise polypeptides added to facilitatepurification and identification of CD40-L (e.g. poly-His), or fusionswith other cytokines to provide novel polyfunctional entities. Othercytokines include, for example, any of interluekins-1 through 13, TNF(tumor necrosis factor), GM-CSF (granulocyte macrophage-colonystimulating factor), G-CSF (granulocyte-colony stimulating factor), MGF(mast cell growth factor), EGF (epidermal growth factor), PDGF(platelet-derived growth factor), NGF (nerve growth factor), EPO(erythropoietin), γ-IFN (gamma interferon), 4-1BB-L (4-1BB ligand) andother cytokines that affect immune cell growth, differentiation orfunction.

Nucleic acid sequences within the scope of the present invention includeDNA and/or RNA sequences that hybridize to the nucleotide sequence ofSEQ ID NO:1 or SEQ ID NO:11 or the complementary strands, underconditions of moderate or severe stringency. Moderate stringencyhybridization conditions refer to conditions described in, for example,Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989). Conditionsof moderate stringency, as defined by Sambrook et al., include use of aprewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) andhybridization conditions of 50° C., 5×SSC, overnight. Conditions ofsevere stringency include higher temperatures of hybridization andwashing.

Biological activity of CD40-L may be determined, for example, bycompetition for binding to the ligand binding domain of CD40 (i.e.competitive binding assays). Both murine CD40-L and human CD40-L bind tohuman CD40. The binding affinity of murine CD40-L (expressed on sortedmurine EL-40.9 cells) for human CD40 was approximately 1.74×10⁹ M⁻¹.Similarly, the binding affinity of murine CD40-L (expressed on unsortedmurine EL-46.1 cells) for human CD40 was approximately 2.3×10⁹ M⁻¹. Bothbinding affinity measurements are within a range typical ofcytokine/cytokine receptor binding.

One configuration of a competitive binding assay for CD40-L polypeptideuses a radiolabeled, soluble murine CD40-L according to FIG. 1 (SEQ IDNO:1) or human CD40-L according to FIG. 2 (SEQ ID NO:11), and intactcells expressing CD40 (e.g., human B cells). Instead of intact cells,one could substitute soluble CD40 (such as a CD40/Fc fusion protein)bound to a solid phase through a Protein A or Protein G interaction withthe Fc region of the fusion protein. A second configuration of acompetitive binding assay utilizes radiolabeled soluble CD40 such as aCD40/Fc fusion protein, and intact cells expressing CD40-L.Alternatively, soluble CD40-L could be bound to a solid phase.

Competitive binding assays can be performed using standard methodology.For example, radiolabeled murine CD40-L can be used to compete with aputative CD40-L homolog to assay for binding activity againstsurface-bound CD40. Qualitative results can be obtained by competitiveautoradiographic plate binding assays, or Scatchard plots may beutilized to generate quantitative results.

Competitive binding assays with intact cells expressing CD40 can beperformed by two methods. In a first method, B cells are grown either insuspension or by adherence to tissue culture plates. Adherent cells canbe removed by treatment with 5 mM EDTA treatment for ten minutes at 37°C. In a second method, transfected COS cells expressing membrane-boundCD40 can be used. COS cells or another mammalian cell can be transfectedwith human CD40 cDNA in an appropriate vector to express full lengthCD40 with an extracellular region exterior to the cell.

Alternatively, soluble CD40 can be bound to a solid phase such as acolumn chromatography matrix, or a tube or similar substrate suitablefor analysis for the presence of a detectable moiety such as ¹²⁵I.Binding to a solid phase can be accomplished, for example, by obtaininga CD40/Fc fusion protein and binding it to a protein A or protein Gsurface.

Another means to measure the biological activity of CD40-L and homologsthereof is to utilize conjugated, soluble CD40 (for example,¹²⁵I-CD40/Fc) in competition assays similar to those described above. Inthis case, however, intact cells expressing CD40-L, or soluble CD40-Lbound to a solid substrate, are used to measure competition for bindingof conjugated, soluble CD40 to CD40-L by a sample containing a putativeCD40 homolog.

CD40-L may also be assayed by measuring biological activity in a B cellproliferation assay. Human B cells may be obtained from human tonsils bypurification by negative selection and Percoll density sedimentation, asdescribed by Defrance et al., J. Immunol. 139:1135, 1987. Burkittlymphoma cell lines may be used to measure cell proliferation inresponse to CD40-L. Examples of Burkitt lymphoma cell lines include, forexample, Raji (ATCC CCL 86), Daudi (ATCC CCL 213) and Namalwa (ATCC CRL1432). Membrane-bound CD40-L stimulated B cell proliferation.Oligomeric, preferably dimeric, CD40-L can stimulate B cellproliferation. CD40 (receptor) antagonizes CD40-L proliferation of Bcells.

Yet another assay for determining CD40-L biological activity is tomeasure immunoglobulin produced by B cells in response to activation byCD40-L or a derivative or analog thereof. Polyclonal immunoglobulinsecretion can be measured, for example, by incubating with 5×10⁵ Bcells/ml in culture for at least seven days. Immunoglobulin (Ig)production can be measured by an ELISA assay such as one described inMaliszewski et al., J. Immunol. 144:3028, 1990 [Maliszewski et al. I] orMaliszewski et al., Eur J. Immunol. 20:1735, 1990 [Maliszewski et al.II]. Murine B cells can be obtained, for example, from mice and culturedaccording to procedures described in Grabstein et al., J. Exp. Med.163:1405, 1986 [Grabstein et al. I], Maliszewski et al. I, andMaliszewski et al. II.

CD40-L can be used in a binding assay to detect cells expressing CD40.For example, murine CD40-L according to FIG. 1 (SEQ ID NO:1) or humanCD40-L according to FIG. 2 (SEQ ID NO:11), or an extracellular domain ora fragment thereof, can be conjugated to a detectable moiety such as¹²⁵I. Radiolabeling with ¹²⁵I can be performed by any of severalstandard methodologies that yield a functional ¹²⁵I-CD40-L moleculelabeled to high specific activity. Alternatively, another detectablemoiety such as an enzyme that can catalyze a colorimetric orfluorometric reaction, biotin or avidin may be used. Cells expressingCD40 can be contacted with conjugated CD40-L. After incubation, unboundconjugated CD40-L is removed and binding is measured using thedetectable moiety.

CD40-L polypeptides may exist as oligomers, such as dimers or trimers.Oligomers are linked by disulfide bonds formed between cysteine residueson different CD40-L polypeptides. Alternatively, one can link twosoluble CD40-L domains with a Gly₄SerGly₅Ser linker sequence, or otherlinker sequence described in U.S. Pat. No. 5,073,627, which isincorporated by reference herein. CD40-L polypeptides may also becreated by fusion of the C terminal of soluble CD40-L (extracellulardomain) to the Fc region of IgG1 (for example, SEQ ID NO:3) as describedfor the CD40/Fc fusion protein. CD40-L/Fc fusion proteins are allowed toassemble much like heavy chains of an antibody molecule to form divalentCD40-L. If fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form a CD40-L oligomer with as many asfour CD40-L extracellular regions.

Fusion proteins can be prepared using conventional techniques of enzymecutting and ligation of fragments from desired sequences. PCR techniquesemploying synthetic oligonucleotides may be used to prepare and/oramplify the desired fragments. Overlapping synthetic oligonucleotidesrepresenting the desired sequences can also be used to prepare DNAconstructs encoding fusion proteins. Fusion proteins can also compriseCD40-L and two or more additional sequences, including a leader (orsignal peptide) sequence, Fc region, linker sequence, and sequencesencoding highly antigenic moieties that provide a means for facilepurification or rapid detection of a fusion protein.

Signal peptides facilitate secretion of proteins from cells. Anexemplary signal peptide is the amino terminal 25 amino acids of theleader sequence of human interleukin-7 (IL-7; Goodwin et al., Proc.Natl. Acad. Sci. U.S.A. 86:302, 1989; FIG. 2B). Other signal peptidesmay also be employed. For example, certain nucleotides in the IL-7leader sequence can be altered without altering the amino acid sequence.Additionally, amino acid changes that do not affect the ability of theIL-7 sequence to act as a leader sequence can be made.

The Flag® octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) does not alterthe biological activity of fusion proteins, is highly antigenic andprovides an epitope reversibly bound by a specific monoclonal antibody,enabling rapid detection and facile purification of the expressed fusionprotein. The Flag® sequence is also specifically cleaved by bovinemucosal enterokinase at the residue immediately following the Asp-Lyspairing, fusion proteins capped with this peptide may also be resistantto intracellular degradation in E. coli. A murine monoclonal antibodythat binds the Flag® sequence has been deposited with the ATCC underaccession number HB 9259; methods of using the antibody in purificationof fusion proteins comprising the Flag® sequence are described in U.S.Pat. No. 5,011,912, which is incorporated by reference herein.

Suitable Fc regions are defined as Fc regions that can bind to protein Aor protein G, or alternatively, are recognized by an antibody that canbe used in purification or detection of a fusion protein comprising theFc region. Preferable Fc regions include the Fc region of human IgG₁ ormurine IgG₁. One example is the human IgG₁ Fc region shown in SEQ IDNO:3; another example is an Fc region encoded by cDNA obtained by PCRfrom oligonucleotide primers from SEQ ID NO:9 and SEQ ID NO:10 withhuman cDNA as a template. Portions of a suitable Fc region may also beused, for example, an Fc region of human IgG₁ from which has beendeleted a sequence of amino acids responsible for binding to protein A,such that the resultant Fc region binds to protein G but not protein A.

The [Gly₄Ser]₃ repeat sequence provides a linker sequence that separatesthe extracellular region of the CD40-L from the Fc portion of the fusionprotein by a distance sufficient to ensure that the CD40-L properlyfolds into its secondary and tertiary structures. Suitable linkersequences (1) will adopt a flexible extended conformation, (2) will notexhibit a propensity for developing an ordered secondary structure whichcould interact with the functional domains of fusion proteins, and (3)will have minimal hydrophobic or charged character which could promoteinteraction with the functional protein domains. Typical surface aminoacids in flexible protein regions include Gly, Asn and Ser. Virtuallyany permutation of amino acid sequences containing Gly, Asn and Serwould be expected to satisfy the above criteria for a linker sequence.Other near neutral amino acids, such as Thr and Ala, may also be used inthe linker sequence. The length of the linker sequence may vary withoutsignificantly affecting the biological activity of the fusion protein.Linker sequences are unnecessary where the proteins being fused havenon-essential N- or C-terminal amino acid regions which can be used toseparate the functional domains and prevent steric interference.

CD40-L polypeptides may exist as soluble polypeptides comprising theextracellular domain of CD40-L as shown in FIG. 1 (SEQ ID NO:1) and FIG.2 (SEQ ID NO:11) or as membrane-bound polypeptides comprising theextracellular domain, a transmembrane region and a short intracellulardomain, as shown in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:11) forthe murine and human sequences, respectively. Moreover, the presentinvention comprises oligomers of CD40-L extracellular domains orfragments thereof, linked by disulfide interactions, or expressed asfusion polymers with or without spacer amino acid linking groups. Forexample, a dimer CD40-L molecule can be linked by an IgG Fc regionlinking group.

Without being bound by theory, membrane-bound CD40-L and oligomericCD40-L can achieve activity stimulating Ig formation and proliferationof B cells previously only achieved by cross-linked anti-CD40 antibodyin the presence of IL-4. It further appears likely that monomericsoluble CD40-L, comprising only the extracellular domain of CD40-L andcapable of binding to CD40 receptor, will serve to antagonize theactivity of membrane-bound and oligomeric CD40-L and/or cross-linkedanti-CD40 antibodies. It further appears likely that the interaction ofmembrane-bound CD40-L with CD40 is the principal molecular interactionresponsible for T cell contact dependent induction of B cell growth anddifferentiation to both antigen specific antibody production andpolyclonal Ig secretion. In this regard, a mammalian cell transfectedwith a cDNA encoding full length CD40-L (i.e., being membrane-bound andhaving an intracellular domain, a transmembrane region and anextracellular domain or a fragment thereof) can mimic T cells in theirability to induce B cell growth, differentiation and stimulation ofantigen-specific antibody production. It appears that activities ofoligomeric soluble CD40-L, preferably an oligomer of extracellularregions, can mimic the biological activities of membrane-bound CD40-L.Moreover, soluble monomeric CD40-L (comprising the extracellular domainor a fragment thereof) can bind to CD40 receptor to prevent T cellinteraction with B cells and therefor have activity similar to CD40(receptor) extracellular domain which itself may be in monomeric or inoligomeric form. Alternatively, CD40-L can be oligomeric to act as asoluble factor capable of inducing B cell growth, differentiation andstimulation of antigen-specific antibody production. Accordingly, itappears that membrane-bound CD40-L and oligomeric CD40-L act as CD40agonists, while soluble (monomeric) CD40-L and soluble CD40 act as CD40antagonists by blocking CD40 receptor sites without significantlytransducing signal or by preventing CD40-L binding to CD40 sites on Bcells and other target cells.

Both CD40 agonists and CD40 antagonists will have useful therapeuticactivity. For example, CD40 agonists (i.e., membrane-bound CD40-L andoligomeric CD40-L) are useful as vaccine adjuvants and for stimulatingmAb production from hybridoma cells. CD40 antagonists (i.e., CD40receptor, CD40/Fc and possibly soluble, monomeric CD40-L) are useful fortreating autoimmune diseases characterized by presence of high levels ofantigen-antibody complexes, such as allergy, lupus, rheumatoidarthritis, insulin dependent diabetes mellitus (IDDM), graft versus hostdisease (GVHD) and others.

IgE secretion from human B cells can be induced by IL-4 in the presenceof T cells (Vercelli et al., J. Exp. Med. 169:1295, 1989). Further, IgEproduction can be induced from T cell depleted PBM (peripheral bloodmononuclear cells) by addition of an anti-CD40 mAb (Jabara et al., J.Exp. Med. 172:1861, 1990 and Zhang et al., J. Immunol. 146:1836, 1991).The present invention further includes a method for inhibiting IgEproduction from activated B cells, activated by IL-4 in the presence ofT cells or by CD40-L (preferably, membrane-bound CD40-L), comprisingadministering an effective amount of a CD40/Fc fusion protein, asdescribed herein, or a soluble CD40 encoded by the cDNA sequencedescribed in SEQ ID NO. 3. Similarly, CD40 receptors and possiblysoluble CD40-L (monomer only) can also block secretion of other antibodyisotypes.

The present invention further includes CD40-L polypeptides with orwithout associated native-pattern glycosylation. CD40-L expressed inyeast or mammalian expression systems (e.g., COS-7 cells) may be similarto or significantly different from a native CD40-L polypeptide inmolecular weight and glycosylation pattern, depending upon the choice ofexpression system. Expression of CD40-L polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules.

DNA constructs that encode various additions or substitutions of aminoacid residues or sequences, or deletions of terminal or internalresidues or sequences not needed for biological activity or binding canbe prepared. For example, the extracellular CD40-L N-glycosylation sitecan be modified to preclude glycosylation while allowing expression of ahomogeneous, reduced carbohydrate analog using yeast expression systems.N-glycosylation sites in eukaryotic polypeptides are characterized by anamino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Yis Ser or Thr. Appropriate modifications to the nucleotide sequenceencoding this triplet will result in substitutions, additions ordeletions that prevent attachment of carbohydrate residues at the Asnside chain. In another example, sequences encoding Cys residues can bealtered to cause the Cys residues to be deleted or replaced with otheramino acids, preventing formation of incorrect intramolecular disulfidebridges upon renaturation. Human CD40-L comprises five Cys residues inits extracellular domain. Thus, at least one of the five Cys residuescan be replaced with another amino acid or deleted without effectingprotein tertiary structure or disulfide bond formation.

Other approaches to mutagenesis involve modification of sequencesencoding dibasic amino acid residues to enhance expression in yeastsystems in which KEX2 protease activity is present. Sub-units of aCD40-L polypeptide may be constructed by deleting sequences encodingterminal or internal residues or sequences. Moreover, other analyses maybe performed to assist the skilled artisan in selecting sites formutagenesis. For example, PCT/US92/03743 (the disclosure of which ishereby incorporated by reference) discusses methods of selecting ligandagonists and antagonists.

CD40-L polypeptides are encoded by multi-exon genes. The presentinvention further includes alternative mRNA constructs which can beattributed to different mRNA splicing events following transcription andwhich share regions of identity or similarity with the cDNAs disclosedherein.

Antisense or sense oligonucleotides comprise a single-stranded nucleicacid sequence (either RNA or DNA) capable of binding to target CD40-LmRNA (sense) or CD40-L DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of SEQ ID NO:1 or SEQ ID NO:11, or a DNA or RNA complement ofSEQ ID NO:1 or SEQ ID NO:11. Such a fragment comprises at least about 14nucleotides. Preferably, such a fragment comprises from about 14 toabout 30 nucleotides. The ability to create an antisense or a senseoligonucleotide, based upon a cDNA sequence for CD40-L is described in,for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krolet al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. Suitable polymerase promoters includepromoters for any RNA polymerase, or promoters for any DNA polymerase.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences. Other examples of sense orantisense oligonucleotides include those oligonucleotides which arecovalently linked to organic moieties, such as those described in WO90/10448, and other moieties that increases affinity of theoligonucleotide for a target nucleic acid sequence, such aspoly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence. Antisense or sense oligonucleotides may be introduced into acell containing the target nucleic acid sequence by any gene transfermethod, including, for example, CaPO₄-mediated DNA transfection,electroporation, or other gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT Application US 90/02656).Alternatively, other promoter sequences may be used to express theoligonucleotide.

Sense or antisense oligonucleotides may also be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

The sequence of murine CD40-L cDNA was obtained by direct expressiontechniques. The sequence of human CD40-L was obtained by cross-specieshybridization techniques using the murine CD40-L cDNA as a probe.

We cloned murine CD40-L by first obtaining a clone of the extracellularregion of human CD40 (the receptor) by polymerase chain reaction (PCR)techniques using primers based upon a sequence published in Stamenkovicet al. (SEQ ID NO:4). An upstream oligonucleotide primer5′-CCGTCGACCACCATGGTTCGTCTGCC-3′ (SEQ ID NO:5) introduces a Sal 1 siteupstream from an initiator methionine of CD40 and a downstreamoligonucleotide primer 5′-CCGTCGACGTCTAGAGCCGATCCTGGGG-3′ (SEQ ID NO:6)inserts a termination codon after amino acid 192 of CD40, followed byXba 1 and Sal 1 sites. The amplified cDNA was digested with Sal 1 andcloned into pDC406 (McMahan et al., EMBO J. 10:2821, 1991) to constructpDC406/s CD40.

A second CD40 receptor fragment (SEQ ID NO:4) was obtained by PCRtechniques for fusion to the Fc domain of human IgG1 (SEQ ID NO:3).Briefly, The upstream oligonucleotide primer (SEQ ID NO:5) and fusiontemplate (SEQ ID NO:4) were the same as before. The downstreamoligonucleotide primer was5′-ACAAGATCTGGGCTCTACGTATCTCAGCCGATCCTGGGGAC-3′ (SEQ ID NO:7) thatinserts amino acids Tyr Val Glu Pro Arg (SEQ ID NO:8) after amino acid193 of CD40. Glu and Pro are the first two amino acids of a hinge regionof human IgG1, and are followed by a Bgl II restriction site. The Bgl IIrestriction site was used to fuse the extracellular domain of CD40 tothe remainder of human IgG1 Fc region.

Other fusion proteins comprising ligand binding domains from otherreceptors can be made by obtaining a DNA sequence for the ligand bindingdomain of a receptor and fusing this sequence to a DNA sequence encodingan Fc region of an antibody molecule that binds to protein A or proteinG, or another polypeptide that is capable of affinity purification, forexample, avidin or streptavidin. The resultant gene construct can beintroduced into mammalian cells to transiently express a fusion protein.Receptor/Fc fusion proteins can be purified by protein A or protein Gaffinity purification. Receptor/avidin fusion proteins can be purifiedby biotin affinity chromatography. The fusion protein can later beremoved from the column by eluting with a high salt solution or anotherappropriate buffer.

We obtained a cDNA encoding human IgG1 Fc region by PCR amplificationusing cDNA from human cells as a template and an upstreamoligonucleotide primer 5′-TATTAATCATTCAGTAGGGCCCAGATCTTGTGACAAAACTCAC-3′(SEQ ID NO:9) and a downstream oligonucleotide primer5′-GCCAGCTTAACTAGTTCATTTACCCGGAGACAGGGAGA-3″ (SEQ ID NO:10). The PCRamplified cDNA introduced a Bgl II site near the beginning of the hingeregion, which was used to ligate CD40 extracellular domain to constructa s CD40/Fc fusion cDNA, which was ligated into pDC406 to constructpDC406/CD40/Fc. Other suitable Fc regions are defined as any region thatcan bind with high affinity to protein A or protein G, and includes theFc region of human IgG1 or murine IgG1. One example is the human IgG1 Fcregion shown in SEQ ID NO:3 or the cDNA obtained by PCR fromoligonucleotide primers from SEQ ID NO:9 and SEQ ID NO:10 with humancDNA as a template.

Receptor/Fc fusion molecules preferably are synthesized in recombinantmammalian cell culture because they are generally too large and complexto be synthesized by prokaryotic expression methods. Examples ofsuitable mammalian cells for expressing a receptor/Fc fusion proteininclude CV-1 cells (ATCC CCL 70) and COS-7 cells (ATCC CRL 1651), bothderived from monkey kidney.

The DNA construct pDC406/CD40/Fc was transfected into the monkey kidneycell line CV-1/EBNA (ATCC CRL 10478). The pDC406 plasmid includesregulatory sequences derived from SV40, human immunodeficiency virus(HIV), and Epstein-Barr virus (EBV). The CV-1/EBNA cell line was derivedby transfection of the CV-1 cell line with a gene encoding Epstein-Barrvirus nuclear antigen-1 (EBNA-1) and constitutively express EBNA-1driven from human CMV immediate-early enhancer/promoter. An EBNA-1 geneallows for episomal replication of expression vectors, such as pDC406,that contain the EBV origin of replication.

Transfectants expressing CD40/Fc fusion protein are initially identifiedusing dot blots or Western blots. The supernatants are then subjected todot blot or gel electrophoresis followed by transfer of theelectrophoresed proteins for binding to G28-5 mAb (an antibody thatbinds to human CD40 receptor). The blotted proteins were then incubatedwith radiolabeled with ¹²⁵I-protein A, washed to remove unbound label,and examined for expression of Fc. Monoclonal antibody G28-5 wasproduced according to Clark et al., supra.

Once cells expressing the fusion construct were identified, large scalecultures of transfected cells were grown to accumulate supernatant fromcells expressing CD40/Fc. CD40/Fc fusion protein in supernatant fluidwas purified by affinity purification. Briefly, one liter of culturesupernatant containing CD40/Fc fusion protein was purified by filteringmammalian cell supernatants (e.g., in a 0.45μ filter) and applyingfiltrate to a protein A/G antibody affinity column (Schleicher andSchuell, Keene, N.H.) at 4° C. at a flow rate of 80 ml/hr for a 1.5cm×12.0 cm column. The column was washed with 0.5 M NaCl in PBS untilfree protein could not be detected in wash buffer. Finally, the columnwas washed with PBS. Bound fusion protein was eluted from the columnwith 25 mM citrate buffer, pH 2.8, and brought to pH 7 with 500 mM Hepesbuffer, pH 9.1. Silver-stained SDS gels of the eluted CD40/Fc fusionprotein showed it to be >98% pure.

Soluble CD40 (sCD40) and CD40/Fc fusion proteins were made as describedherein. The supernatants were purified through a G28-5 (anti-CD40 mAb)affinity column to affinity purify sCD40 expressed by the transfectedCV-1/EBNA cells. Protein-containing fractions were pooled and aliquotsremoved for G28-5 binding assays and analysis by SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) in the presence of 1mM dithiothreitol as a reducing agent. A single band was seen ofmolecular weight 28,100 daltons. In the absence of a reducing agent,SDS-PAGE analysis of sCD40 revealed two bands, a major band of molecularweight 56,000 and a minor band of molecular weight 28,000. The bandingpattern indicates that the majority of sCD40 exists as adisulfide-linked homodimer in solution. The 28,000 band is free monomer.

CD40 proteins were visualized by silver staining. Sample proteinconcentrations were determined using a micro-BCA assay (Pierce) withultrapure bovine serum albumin as standard. Soluble CD40 purity andprotein concentration were confirmed by amino acid analysis. Purifiedsoluble CD40 was absorbed to PVDF paper and the paper subjected toautomated Edman degradation on an Applied Biosystems model 477A proteinsequencer according to manufacturers instructions for N-terminal proteinsequencing. This procedure checked the protein sequence of sCD40.

Soluble CD40 and CD40/Fc fusion protein were able to modulate human Bcell responses in the absence of anti-CD40 mAb (G28-5). Purifiedtonsillar B cells were cultured with anti-IgM and human IL-4 and eithersCD40 or CD40/Fc fusion protein was added. Neither form of CD40 had aninhibitory effect on B cell proliferation (as measured by tritiatedthymidine incorporation). IL-4 receptor, by contrast, inhibitedIL-4-induced B cell proliferation in a concentration-dependent manner.

Soluble CD40 and CD40/Fc were tested for their ability to inhibit IL-4induced IgE secretion in a 2-donor MLC (mixed lymphocyte culture)system. In three experiments, the level of IgE production was reduced asthe concentration of CD40 was increased. Soluble CD40, added at aconcentration of 10 μg/ml, was able to completely inhibit IgE secretionin this model of allergy. Further, CD40/Fc had similar effects as itssoluble counterpart. However, addition of an IL-7 receptor-Fc fusionprotein (made by similar procedures with a published IL-7 receptorsequence) did not affect secretion of IgE in this model.

Levels of CD23 were also measured in the same MLC in response to sCD40or CD40/Fc fusion proteins. Soluble CD40 produced a small, butreproducible decrease in sCD23 level at day 6 compared to culturesstimulated with IL-4 alone, however a stronger inhibitory effect waspronounced at day 12 in the same cultures. Soluble CD23 induction byIL-4-stimulated T-depleted PBM (peripheral blood macrophages) E⁻ cellswas similarly affected by addition of sCD40, causing a small decrease insCD23 levels at day 6 and a more pronounced inhibition at day 12. Ineach culture system, the results with CD40/Fc fusion protein weresubstantially the same as with sCD40.

In an effort to isolate a cDNA for a CD40-L, purified CD40/Fc fusionprotein was radioiodinated with ¹²⁵I using a commercially availablesolid phase agent (IODO-GEN, Pierce). In this procedure, 5 μg ofIODO-GEN were plated at the bottom of a 10×75 mm glass tube andincubated for twenty minutes at 4° C. with 75 μl of 0.1 M sodiumphosphate, pH 7.4 and 20 μl (2 mCi) Na¹²⁵I. The solution was thentransferred to a second glass tube containing 5 μg of CD40/Fc in 45 μlPBS (phosphate buffered saline) and this reaction mixture was incubatedfor twenty minutes at 4° C. The reaction mixture was fractionated by gelfiltration on a 2 ml bed volume of Sephadex® G-25 (Sigma), and thenequlibrated in RPMI 1640 medium containing 2.5% (v/v) bovine serumalbumin (BSA), 0.2% (v/v) sodium azide and 20 mM Hepes, pH 7.4 bindingmedium. The final pool of ¹²⁵I CD40/Fc was diluted to a working stocksolution of 1×10⁻⁷ M in binding medium and stored for up to one month at4° C. without detectable loss of receptor binding activity.

A cDNA library was prepared from a EL4 cell line sorted by FACS(fluorescence activated cell sorting) on the basis of binding of abiotinylated CD40/Fc fusion protein. Cells were sorted five times untilthere was a significant shift in fluorescence intensity based uponexpression of a ligand for CD40 by the sorted EL-4 cells. The five-timessorted cells were called EL-40.5 cells and these cells were cultured forthe purposes of creating a cDNA library from EL-40.5 mRNA. Briefly, cDNAwas synthesized, inserted into empty pDC406 vector and transformed intoE. coli. Transformants were pooled, and the DNA from the pools wasisolated and transfected into CV1-EBNA cells to create an expressioncloning library. Transfected CV1-EBNA cells were cultured on slides forthree days to permit transient expression of CD40-L. The slidescontaining the transfected cells were then incubated with radioiodinatedCD40/Fc, washed to remove unbound CD40/Fc, and fixed withgluteraldehyde. The fixed slides were dipped in liquid photographicemulsion and exposed in the dark. After developing the slides, they wereindividually examined with a microscope and cells expressing CD40-L wereidentified by the presence of autoradiographic silver grains against alight background.

The expression cloning library from EL-40.5 cells was screened and onepool, containing approximately 2000 individual clones, was identified aspositive for binding ¹²⁵I labeled CD40/Fc fusion protein. This pool wasbroken down into smaller pools of approximately 200 colonies. Thesmaller pools were screened as described above. One of the smaller poolswas positive for CD40-L.

A single clone was isolated and sequenced by standard techniques, toprovide the cDNA sequence and deduced amino acid sequence of murineCD40-L as shown in FIG. 1 and SEQ ID NO:1.

The human homolog CD40-L cDNA was found by cross species hybridizationtechniques. Briefly, a human peripheral blood lymphocyte (PBL) cDNAlibrary was made from peripheral blood lymphocytes treated with OKT3antibody (ATCC, Rockville Md.) that binds to CD3 (10 ng/ml) andinterleukin-2 (IL-2, 10 ng/ml) for six days. The PBL cells were washedand then stimulated for 4 hours with 10 ng/ml PMA (phorbol myristateacetate, Sigma St Louis) and 500 ng/ml ionomycin (Calbiochem). MessengerRNA was isolated from stimulated PBL cells, cDNA formed and cDNA wasligated into Eco R1 linkers. Ligated cDNA was inserted into the Eco R1site of λgt10 phage cloning vehicle (Gigapak® Stratagene, San Diego,Calif.) according to manufacturer's instructions. Phage were amplified,plated at densities densities of approximately 20,000 phage per 15 cmplate, and phage lifts were performed, as described in Maniatis et al.,Molecular Biology: A Laboratory Manual, Cold Spring Harbor Laboratory,NY, 1982, pages 316-328. A murine probe was constructed corresponding tothe coding region of murine CD40-L from nucleotide 13 to nucleotide 793of SEQ ID NO:1 and FIG. 1. This probe was hybridized to to the PBLlibrary phage lifts under conditions of moderate to severe stringency.Briefly, hybridization conditions were 6×SSC, 1×Denhardt's solution, 2mM EDTA, 0.5% NP40 (Nonidet P-40 detergent) at 63° C. overnight. Thiswas followed by washing in 3×SSC, 0.1% SDS for three hours at 55° C.,followed by overnight exposure to X-Ray film. Positive plaques wereidentified at a frequency of approximately 1 per 1000 plaques. Positiveplaques were purified twice and cDNA was prepared from amplifiedcultures.

One can utilize the murine or human CD40-L cDNA sequences disclosedherein to obtain cDNAs encoding other mammalian homologs of murine orhuman CD40-L by cross-species hybridization techniques. Briefly, anoligonucleotide probe is created from the nucleotide sequence of theextracellular region of murine CD40-L as described in FIG. 1 (SEQ IDNO:1) or human CD40-L as described in FIG. 2 (SEQ ID NO:11). This probecan be made by standard techniques, such as those described in Maniatiset al. supra. The murine or human probe is used to screen a mammaliancDNA library or genomic library under moderate stringency conditions.Examples of mammalian cDNA or genomic libraries include, for cDNA, alibrary made from the mammal's peripheral blood lymphocytes.Alternatively, various cDNA libraries or mRNAs isolated from variouscell lines can be screened by Northern hybridization to determine asuitable source of mammalian CD40-L DNA or mRNA.

Recombinant expression vectors for expression of CD40-L by recombinantDNA techniques include a CD40-L DNA sequence comprising a synthetic orcDNA-derived DNA fragment encoding a CD40-L polypeptide, operably linkedto a suitable transcriptional or translational regulatory nucleotidesequence, such as one derived from a mammalian, microbial, viral, orinsect gene. Examples of regulatory sequences include sequences having aregulatory role in gene expression (e.g., a transcriptional promoter orenhancer), optionally an operator sequence to control transcription, asequence encoding an mRNA ribosomal binding site, and appropriatesequences which control transcription and translation initiation andtermination. Nucleotide sequences are operably linked when theregulatory sequence functionally relates to the CD40-L DNA sequence.Thus, a promoter nucleotide sequence is operably linked to a CD40-L DNAsequence if the promoter nucleotide sequence controls the transcriptionof the CD40-L DNA sequence. Still further, a ribosome binding site maybe operably linked to a sequence for a CD40-L polypeptide if theribosome binding site is positioned within the vector to encouragetranslation. In addition, sequences encoding signal peptides can beincorporated into expression vectors. For example, a DNA sequence for asignal peptide (secretory leader) may be operably linked to a CD40-L DNAsequence. The signal peptide is expressed as a precursor amino acidsequence which enables improved extracellular secretion of translatedfusion polypeptide by a yeast host cell.

Suitable host cells for expression of CD40-L polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gramnegative or gram positive organisms, for example, E. coli or Bacilli.Suitable prokaryotic host cells for transformation include, for example,E. coli, Bacillus subtilis, Salmonella typhimurium, and various otherspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus.Higher eukaryotic cells include established cell lines of mammalianorigin. Cell-free translation systems could also be employed to produceCD40-L polypeptides using RNAs derived from DNA constructs disclosedherein. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are described,for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual,Elsevier, New York, (1985).

In a prokaryotic host cell, such as E. coli, a CD40-L polypeptide oranalog may include an N-terminal methionine residue to facilitateexpression of the recombinant polypeptide in the prokaryotic host cell.The N-terminal Met may be cleaved from the expressed recombinant CD40-Lpolypeptide. Prokaryotic host cells may be used for expression of CD40-Lpolypeptides that do not require extensive proteolytic or disulfideprocessing.

The expression vectors carrying the recombinant CD40-L DNA sequence aretransfected or transformed into a substantially homogeneous culture of asuitable host microorganism or mammalian cell line. Transformed hostcells are cells which have been transformed or transfected withnucleotide sequences encoding CD40-L polypeptides and express CD40-Lpolypeptides. Expressed CD40-L polypeptides will be located within thehost cell and/or secreted into culture supernatant fluid, depending uponthe nature of the host cell and the gene construct inserted into thehost cell.

Expression vectors transfected into prokaryotic host cells generallycomprise one or more phenotypic selectable markers. A phenotypicselectable marker is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement, and an origin of replication recognized by the host toensure amplification within the host. Other useful expression vectorsfor prokaryotic host cells include a selectable marker of bacterialorigin derived from commercially available plasmids. This selectablemarker can comprise genetic elements of the cloning vector pBR322 (ATCC37017). pBR322 contains genes for ampicillin and tetracycline resistanceand thus provides simple means for identifying transformed cells. ThepBR322 “backbone” sections are combined with an appropriate promoter anda CD40-L DNA sequence. Other commercially vectors include, for example,pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (PromegaBiotec, Madison, Wis., USA).

Promoter sequences are commonly used for recombinant prokaryotic hostcell expression vectors. Common promoter sequences include β-lactamase(penicillinase), lactose promoter system (Chang et al., Nature 275:615,1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp)promoter system (Goeddel et al., Nucl. Acads Res. 8:4057, 1980; andEP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularlyuseful prokaryotic host cell expression system employs a phage λ P_(L)promoter and a cI857ts thermolabile repressor sequence. Plasmid vectorsavailable from the American Type Culture Collection which incorporatederivatives of the λ P_(L) promoter include plasmid pHUB2 (resident inE. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1(ATCC 53082)).

CD40-L may be expressed in yeast host cells, preferably from theSaccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, suchas Pichia or Kluyveromyces, may also be employed. Yeast vectors willoften contain an origin of replication sequence from a 2μ yeast plasmid,an autonomously replicating sequence (ARS), a promoter region, sequencesfor polyadenylation, and sequences for transcription termination.Preferably, yeast vectors include an origin of replication sequence andselectable marker. Suitable promoter sequences for yeast vectors includepromoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman etal., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess etal., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.17:4900, 1978), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657.

Yeast vectors can be assembled, for example, using DNA sequences frompBR322 for selection and replication in E. coli (Amp^(r) gene and originof replication). Other yeast DNA sequences that can be included in ayeast expression construct include a glucose-repressible ADH2 promoterand α-factor secretion leader. The ADH2 promoter has been described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). The yeast α-factor leader sequence directs secretion ofheterologous polypeptides. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant CD40-L polypeptides. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines. Suitable mammalian expression vectorsinclude nontranscribed elements such as an origin of replication, apromoter sequence, an enhancer linked to the structural gene, other 5′or 3′ flanking nontranscribed sequences, such as ribosome binding sites,a polyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. For example,commonly used mammalian cell promoter sequences and enhancer sequencesare derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40),and human cytomegalovirus. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early and late promoter, enhancer,splice, and polyadenylation sites may be used to provide the othergenetic elements required for expression of a structural gene sequencein a mammalian host cell. Viral early and late promoters areparticularly useful because both are easily obtained from a viral genomeas a fragment which may also contain a viral origin of replication(Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragmentsmay also be used, provided the approximately 250 bp sequence extendingfrom the Hind III site toward the Bgl I site located in the SV40 viralorigin of replication site is included.

Exemplary mammalian expression vectors can be constructed as disclosedby Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful highexpression vector, PMLSV N1/N4, described by Cosman et al., Nature312:768, 1984 has been deposited as ATCC 39890. Additional usefulmammalian expression vectors are described in EP-A-0367566, and in U.S.patent application Ser. No. 07/701,415, filed May 16, 1991, incorporatedby reference herein. For expression of a type II protein extracellularregion, such as CD40-L, a heterologous signal sequence should be added,such as the signal sequence for interleukin-7 (IL-7) described in U.S.Pat. No. 4,965,195, or the signal sequence for interleukin-2 receptordescribed in U.S. patent application Ser. No. 06/626,667 filed on Jul.2, 1984.

Human or murine CD40-L can be made in membrane-bound form when anintracellular and transmembrane regions are included or in soluble formwith only the extracellular domain. We expressed full length murineCD40-L in mammalian cells to yield cells expressing membrane-boundmurine CD40-L. CV1 cells were transfected with a cDNA shown in FIG. 1(SEQ ID NO:1) in HAVEO vector or CV1 cells were transfected with HAVEOempty vector using techniques described in Example 6 herein. Thisyielded transfected CV1 cells expressing membrane-bound murine CD40-L.These cells were used as a source of membrane-bound murine CD40-L forthe series of experiments reported in Examples 10-13 reported below.

Purification of Recombinant CD40-L Polypeptides

CD40-L polypeptides may be prepared by culturing transformed host cellsunder culture conditions necessary to express CD40-L polypeptides. Theresulting expressed polypeptides may then be purified from culture mediaor cell extracts. A CD40-L polypeptide, if desired, may be concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify CD40-L. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous recombinant protein.

It is also possible to utilize an affinity column comprising CD40 ligandbinding domain to affinity-purify expressed CD40-L polypeptides. CD40-Lpolypeptides can be removed from an affinity column in a high saltelution buffer and then dialyzed into a lower salt buffer for use.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Transformed yeast host cells are preferably employed to express CD40-Las a secreted polypeptide. This simplifies purification. Secretedrecombinant polypeptide from a yeast host cell fermentation can bepurified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 296:171, 1984). Urdal et al. describe two sequential,reversed-phase HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column.

Administration of CD40-L Compositions

The present invention provides therapeutic compositions comprising aneffective amount of CD40-L in a suitable diluent or carrier and methodsof treating mammals using the compositions. For therapeutic use,purified CD40-L or a biologically active analog thereof is administeredto a patient, preferably a human, for treatment in a manner appropriateto the indication. Thus, for example, CD40-L pharmaceutical compositions(for example, in the form of a soluble extracellular domain, or afragment thereof) which is administered to achieve a desired therapeuticeffect can be given by bolus injection, continuous infusion, sustainedrelease from implants, or other suitable technique. Typically, a CD40-Ltherapeutic agent will be administered in the form of a pharmaceuticalcomposition comprising purified CD40-L polypeptide in conjunction withphysiologically acceptable carriers, excipients or diluents. Suchcarriers will be nontoxic to patients at the dosages and concentrationsemployed. Ordinarily, the preparation of such compositions entailscombining a CD40-L polypeptide with buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose or dextrans, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.CD40-L sense or antisense oligonucleotides may be administered in vivoby administering an effective amount of a vector containing a nucleicacid sequence that encodes and effective antisense or senseoligonucleotide. Additionally, CD40-L sense or antisenseoligonucleotides may be administered ex vivo by removing cellscontaining CD40-L DNA or mRNA from an individual, incorporating anantisense or sense oligonucleotide into the cells using gene transfertechniques, and re-infusing the cells into the individual.

The following examples are intended to illustrate particular embodimentsand not limit the scope of the invention.

EXAMPLE 1

This example describes construction of a CD40/Fc DNA construct toexpress a soluble CD40/Fc fusion protein for use in detecting cDNAclones encoding a CD40 ligand. The cDNA sequence of the extracellularregion or ligand binding domain of complete CD40 human receptor sequencewas obtained using polymerase chain reaction (PCR) techniques, and isbased upon the sequence published in Stamenkovic et al., supra. A CD40plasmid (CDM8) was used as a template for PCR amplification. CDM8 isdescribed in Stamenkovic et al. and was obtained from the authors. A PCRtechnique (Sarki et al., Science 239:487, 1988) was employed using 5′(upstream) and 3′ (downstream) oligonucleotide primers to amplify theDNA sequences encoding CD40 extracellular ligand binding domain.Upstream oligonucleotide primer 5′-CCGTCGACCACCATGGTTCGTCTGCC-3′ (SEQ IDNO:5) introduces a Sal I site upstream from an initiator methionine ofCD40 and a downstream oligonucleotide primer5′-ACAAGATCTGGGCTCTACGTATCTCAGCCGATCCTGGGGAC-3′ (SEQ ID NO:7) thatinserts amino acids Tyr Val Glu Pro Arg (SEQ ID NO:8) after amino acid193 of CD40. Glu and Pro are the first two amino acids of a hinge regionof human IgG1, and are followed by a Bgl II restriction site that wasused to fuse the extracellular domain of CD40 to the remained of humanIgG1 Fc region.

The DNA construct pDC406/CD40/Fc was transfected into the monkey kidneycell line CV-1/EBNA (ATCC CRL 10478). The pDC406 plasmid includesregulatory sequences derived from SV40, human immunodeficiency virus(HIV), and Epstein-Barr virus (EBV). The CV-1/EBNA cell line was derivedby transfection of the CV-1 cell line with a gene encoding Epstein-Barrvirus nuclear antigen-1 (EBNA-1) that constitutively expresses EBNA-1driven from the human CMV intermediate-early enhancer/promoter. TheEBNA-1 gene allows for episomal replication of expression vectors, suchas pDC406, that contain the EBV origin of replication.

Once cells expressing the fusion construct were identified, large scalecultures of transfected cells were grown to accumulate supernatant fromcells expressing CD40/Fc. The CD40/Fc fusion protein in supernatantfluid was purified by affinity purification. Briefly, one liter ofculture supernatant containing the CD40/Fc fusion protein was purifiedby filtering mammalian cell supernatants (e.g., in a 0.45μ filter) andapplying filtrate to a protein A/G antibody affinity column (Schleicherand Schuell, Keene, N.H.) at 4° C. at a flow rate of 80 ml/hr for a 1.5cm×12.0 cm column. The column was washed with 0.5 M NaCl in PBS(phosphate buffered saline) until free protein could not be detected inwash buffer. Finally, the column was washed with PBS. Bound fusionprotein was eluted from the column with 25 mM citrate buffer, pH 2.8,and brought to pH 7 with 500 mM Hepes buffer, pH 9.1. Silver-stained SDSgels of the eluted CD40/Fc fusion protein showed it to be >98% pure.

Purified CD40/Fc fusion protein was iodinated with ¹²⁵I using acommercially available solid phase agent (IODO-GEN, Pierce). In thisprocedure, 5 μg of IODO-GEN were plated at the bottom of a 10×75 mmglass tube and incubated for twenty minutes at 4° C. with 75 μl of 0.1 Msodium phosphate, pH 7.4 and 20 μl (2 mCi) Na¹²⁵I. The solution was thentransferred to a second glass tube containing 5 μg of CD40/Fc in 45 μlPBS and this reaction mixture was incubated for twenty minutes at 4° C.The reaction mixture was fractionated by gel filtration on a 2 ml bedvolume of Sephadex® G-25 (Sigma), and then equlibrated in RPMI 1640medium containing 2.5% (v/v) bovine serum albumin (BSA), 0.2% (v/v)sodium azide and 20 mM Hepes, pH 7.4 binding medium. The final pool of¹²⁵I CD40/Fc was diluted to a working stock solution of 1×10⁻⁷ M inbinding medium and stored for up to one month at 4° C. withoutdetectable loss of receptor binding activity.

Approximately 50%-60% label incorporation was observed. Radioiodinationyielded specific activities in the range of 1×10¹⁵ to 5×10¹⁵ cpm/nmole(0.42-2.0 atoms of radioactive iodine per molecule of protein). SDSpolyacrylamide gel electrophoresis (SDS-PAGE) revealed a single labeledpolypeptide consistent with expected values. The labeled fusion proteinwas greater than 98% trichloroacetic acid (TCA) precipitable, indicatingthat the ¹²⁵I was covalently bound to the protein.

EXAMPLE 2

This example describes selection of a cell line putatively expressingCD40-L. Several cell lines were screened using the radioioidinatedCD40/Fc fusion protein described in Example 1. Briefly, quantitativebinding studies were performed according to standard methodology, andScatchard plots were derived for the various cell lines. A clonal cellline (EL4, ATCC Catalog TIP 39) a murine thymoma cell line wasidentified and sorted. Prior to sorting, EL-4 cells were found toexpress approximately 450 molecules of CD40-L per cell. The seventh sortcells were called EL-40.7 and were grown and found to expressapproximately 10,000 molecules of CD40-L per cell. Lastly, the ninthsort cells were called EL-40.9 and were grown and found to expressapproximately 15,000 molecules of CD40-L per cell.

EXAMPLE 3

This example describes preparation of a cDNA library for expressioncloning of murine CD40-L. The library was prepared from a fifth sortedclone of a mouse thymoma cell line EL-4 (ATCC TIB 39), called EL-40.5.EL-40.5 cells were EL4 cells sorted five times with biotinylated CD40/Fcfusion protein in a FACS (fluorescence activated cell sorter). A cDNAlibrary was made from RNA obtained from EL-40.5 cells essentially asdescribed in U.S. Pat. No. 4,968,607, the disclosure of which isincorporated by reference herein. Briefly, a cDNA library wasconstructed by reverse transcription of poly (A)⁺ mRNA isolated from thetotal RNA extracted from the EL-40.5 cell line. The library constructiontechnique was substantially similar to that described by Ausubel et al.,eds., Current Protocols In Molecular Biology, Vol. 1, (1987). Poly (A)⁺mRNA was isolated by oligo dT cellulose chromatography anddouble-stranded cDNA was made substantially as described by Gubler etal., Gene 25:263, 1983. Poly(A)⁺ mRNA fragments were converted toRNA-cDNA hybrids by reverse transcriptase using random hexanucleotidesas primers. The RNA-cDNA hybrids were then converted intodouble-stranded cDNA fragments using RNAase H in combination with DNApolymerase I. The resulting double-stranded cDNA was blunt-ended with T4DNA polymerase.

     Sal I adaptors 5′- TCG ACT GGA ACG AGA CGA CCT GCT -3′          GACCT TGC TCT GCT GGA CGA - 5′were ligated to 5′ ends of resulting blunt-ended cDNA, as described inHaymerle et al., Nucleic Acids Res. 14:8615, 1986. Non-ligated adaptorswere removed by gel filtration chromatography at 68° C. This left 24nucleotide non-self-complementary overhangs on cDNA. The same procedurewas used to convert 5′ Sal I ends of the mammalian expression vectorpDC406 to 24 nucleotide overhangs complementary to those added to cDNA.Optimal proportions of adaptored vector and cDNA were ligated in thepresence of T4 polynucleotide kinase. Dialyzed ligation mixtures wereelectroporated into E. coli strain DH5α and transformants selected onampicillin plates.

Plasmid DNA was isolated from pools consisting of approximately 2,000clones of transformed E. coli per pool. The isolated DNA was transfectedinto a sub-confluent layer of CV1-EBNA cells using DEAE-dextran followedby chloroquine treatment substantially according to the proceduresdescribed in Luthman et al., Nucl. Acids Res. 11:1295, 1983 andMcCutchan et al., J. Natl. Cancer Inst. 41:351, 1986.

CV1-EBNA cells were maintained in complete medium (Dulbecco's modifiedEagles' media containing 10% (v/v fetal calf serum, 50 U/ml penicillin,50 U/ml streptomycin, and 2 mM L-glutamine) and were plated to a densityof approximately 2×10⁵ cells/well in single-well chambered slides(Lab-Tek). The slides were pre-treated with 1 ml human fibronectin (10μg/ml PBS) for 30 minutes followed by a single washing with PBS. Mediawas removed from adherent cells growing in a layer and replaced with 1.5ml complete medium containing 66.6 μM chloroquine sulfate. About 0.2 mlof a DNA solution (2 μg DNA, 0.5 mg/ml DEAE-dextran in complete mediumcontaining chloroquine) was added to the cells and the mixture wasincubated at 37° C. for about five hours. Following incubation, mediawas removed and the cells were shocked by addition of complete mediumcontaining 10% DMSO (dimethylsulfoxide) for 2.5-20 minutes. Shocking wasfollowed by replacement of the solution with fresh complete medium. Thecells were grown in culture for two to three days to permit transientexpression of the inserted DNA sequences. These conditions led to a 30%to 80% transfection frequency in surviving CV1-EBNA cells.

EXAMPLE 4

This example describes screening of the expression cloning library madein Example 3 with a labeled CD40/Fc fusion protein made in Example 1.After 48-72 hours, transfected monolayers of CV1-EBNA cells made inExample 3 were assayed by slide autoradiography for expression of CD40-Lusing radioiodinated CD40/Fc fusion protein as prepared in Example 1.Transfected CV1-EBNA cells were washed once with binding medium (RPMI1640 containing 25 mg/ml bovine serum albumin (BSA), 2 mg/ml sodiumazide, 20 mM Hepes pH 7.2, and 50 mg/ml nonfat dry milk) and incubatedfor 2 hours at 4° C. in binding medium containing 1×10⁻⁹ M ¹²⁵I-CD40/Fcfusion protein. After incubation, cells in the chambered slides werewashed three times with binding buffer, followed by two washes with PBS,(pH 7.3) to remove unbound radiolabeled fusion protein.

The cells were fixed by incubating in 10% gluteraldehyde in PBS (30minutes at room temperature), washed twice in PBS and air-dried. Theslides were dipped in Kodak GTNB-2 photographic emulsion (6×dilution inwater) and exposed in the dark for two to four days days at roomtemperature in a light-proof box. The slides were developed in Kodak D19developer, rinsed in water and fixed in Agfa G433C fixer. The slideswere individually examined under a microscope at 25-40×magnification.Positive slides showing cells expressing CD40-L were identified by thepresence of autoradiographic silver grains against a light background.

Once pool containing approximately 2000 individual clones was identifiedas potentially positive for binding the CD40/Fc fusion protein. The poolwas titered and plated to provide plates containing approximately 200colonies each. Each plate was scraped to provide pooled plasmid DNA fortransfection into CV1-EBNA cells according to the same proceduredescribed above. The smaller pools were screened by slideautoradiography as described previously. One of the smaller poolscontained clones that were positive for CD40-L as indicated by thepresence of an expressed gene product capable of binding to the CD40/Fcfusion protein.

The positive smaller pool was titered and plated to obtain individualcolonies. Approximately 400 individual colonies were picked andinoculated into culture medium in individual wells of 96-well plates.Cultures were mixed by pooling rows and columns and the mixed cultureswere used to prepare DNA for a final round of transfection andscreening. An intersection of a positive row and and a positive columnindicated a potential positive colony. Ten potential positive colonies(i.e., candidate clones) were identified. DNA was isolated from eachcandidate clone, retransfected and rescreened. Five candidate cloneswere positive by binding to CD40/Fc. All five positive candidate clonescontained a cDNA insert of 1468 nucleotides, as determined bydideoxynucleotide sequencing. The cDNA coding region of the CD40-L clonecorresponds to the sequence of FIG. 1 and SEQ ID NO:1.

A cloning vector containing murine CD40-L sequence, designatedpDC406-mCD40-L, was deposited with the American Type Culture Collection,Rockville, Md. (ATCC) on Dec. 6, 1991, under accession number 68872. Thenucleotide sequence and predicted amino acid sequence of this clone areillustrated in SEQ ID NO:1 and in FIG. 1.

EXAMPLE 5

This example illustrates a cross-species hybridization technique whichwas used to isolate a human CD40-L homolog using a probe designed fromthe sequence of murine CD40-L. A murine CD40-L probe was produced byexcising the coding region from murine CD40-L clone pDC406-CD40-L(nucleotide 13 through 793) and ³²P-labeling the fragment using randomprimers (Boehringer-Mannheim).

A human peripheral blood lymphocyte (PBL) cDNA library was constructedin a λ phage vector using λgt10 arms and packaged in vitro using acommercially available kit (Gigapak® Stratagene, San Diego, Calif.)according to the manufacturer's instructions. The PBL cells wereobtained from normal human volunteers and treated with 10 ng/ml of OKT3(an anti-CD3 antibody), and 10 ng/ml of human IL-2 (Immunex, Seattle,Wash.) for six days. The PBL cells were washed and stimulated with 500ng/ml ionomycin (Calbiochem) and 10 ng/ml PMA (Sigma) for four hours.Messenger RNA and cDNA were obtained from the stimulated PBL cells andpackaged into λgt10 phage vectors (Gigapak® Stratagene) according tomanufacturer's instructions.

The murine probe was hybridized to phage cDNA in 6×SSC (15 mM trisodiumcitrate, and 165 mM sodium chloride), 1×Denhardt's solution, 2 mM EDTA,0.5% Np40 at 63° C. overnight. Hybridization was followed by extensivewashing in 3×SSC, 0.1% SDS at approximately 55° C. for three hours.Specific bands were visualized by autoradiography.

A cloning vector containing human CD40-L sequence, designated hCD40-L,was deposited with the American Type Culture Collection, Rockville, Md.(ATCC) on Dec. 6, 1991, under accession number 68873. The nucleotidesequence and predicted amino acid sequence of this clone are illustratedSEQ ID NO:11 and in FIG. 2.

EXAMPLE 6

This example illustrates the expression of membrane-bound murine CD40-Lin CV1-EBNA cells. Murine CD40-L cDNA in HAVEO vector or empty HAVEOvector were transfected into CV1 EBNA cells using standard techniques,such as those described in McMahan et al. et al. EMBO J. 10:2821, 1991and in Example 3 herein. Briefly, CV1 EBNA cells were plated at adensity of 2×10⁶ cells per 10 cm dish in 10 ml of Dulbecco's MinimalEssential Medium supplemented with 10% fetal calf serum (Medium). Thecells were allowed to adhere overnight at 37° C. The Medium was replacedwith 1.5 ml of Medium containing 66.7 μM chloroquine and a DNA mixturecontaining 5 μg of cDNA encoding mCD40-L. Medium containing 175 μl, and25 μl of DEAE dextran (4 mg/ml in PBS) was also added to the cells. Thecells and cDNA were incubated at 37° C. for 5 hours. The cDNA mixturewas removed and the cells were shocked with 1 ml of fresh Mediumcontaining 10% DMSO for 2.5 min. The Medium was replaced with freshMedium and the cells were grown for at least 3 days.

EXAMPLE 7

This example illustrates the preparation of monoclonal antibodies toCD40-L. Preparations of purified murine CD40-L or human CD40-L areprepared by COS cell expression and CD40/Fc affinity purification asdescribed herein. Purified CD40-L or transfected cells expressingmembrane-bound CD40-L can generate monoclonal antibodies against CD40-Lusing conventional techniques, for example, those techniques describedin U.S. Pat. No. 4,411,993. Briefly, mice are immunized with humanCD40-L as an immunogen emulsified in complete Freund's adjuvant oranother suitable adjuvant such as incomplete Freund's adjuvant or Ribiadjuvant R700 (Ribi, Hamilton, Mont.) or another suitable adjuvant, andinjected in amounts ranging from 10-100 μg subcutaneously orintraperitoneally. Rats (i.e. Lewis rats) are immunized with murineCD40-L as an immunogen in a similar manner. Ten days to three weekslater, the immunized animals are boosted with additional CD40-Lemulsified in incomplete Freund's adjuvant. Mice are periodicallyboosted thereafter on a weekly, bi-weekly or every third weekimmunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision for testing by dot blotassay, ELISA (Enzyme-Linked Immunosorbent Assay), or FACS analysis, forCD40-L antibodies.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of CD40-L in saline. Threeto four days later, the animals are sacrificed, spleen cells harvested,and spleen cells are fused to a murine myeloma cell line (e.g., NS1 orAg 8.653). Fusions generate hybridoma cells, which are plated inmultiple microtiter plates in a selective medium containing HAT(hypoxanthine, aminopterin and thymidine) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and spleen cell-spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified CD40-L by adaptations of the techniques disclosed in Engvall etal., Immunochem. 8:871, 1971 and in U.S. Pat. No. 4,703,004, or by orFACS as described herein. Positive hybridoma cells can be cloned in softagar or another, similar medium, or by limiting dilution, to insure thata final cell population is derived from a single hybridoma cell. Thecloned hybridoma cells are injected intraperitoneally into syngeneicmice (or rats) to produce ascites containing high concentrations ofanti-CD40-L monoclonal antibodies. Alternatively, hybridoma cells can begrown in vitro in flasks or roller bottles by various techniques.Monoclonal antibodies produced in mouse ascites can be purified byammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can also be used, as canaffinity chromatography based upon binding to CD40-L.

EXAMPLE 8

This example illustrates anti-allergy therapeutic effects of sCD40 andCD40/Fc fusion protein. Soluble CD40 and CD40/Fc were tested for theirability to inhibit IL-4 (5 ng/ml) induced IgE secretion in a two donorMLC system. The data from three experiments are presented in Table 1.

TABLE 1 IgE (ng/ml) Addition Exp. 1 Exp. 2 Exp. 3 medium <0.1 <0.1 <0.1IL-4 24 47 54 IL-4 + sCD40 (0.1 μg/ml) 19 nd 38 IL-4 + sCD40 (0.3 μg/ml)14 29 24 IL-4 + sCD40 (1 μg/ml) 10 24 8 IL-4 + sCD40 (3 μg/ml) 7 19 2IL-4 + IL-7R/Fc (10 μg/ml) 21 nd 58

IgE levels were measured after 12 days in culture by an ELISA procedure.Briefly, flat-bottomed 96-well microtiter plates (Corning) were coatedwith mouse mAb anti-human IgE (Zymed) at 1:500 dilution in PBS(phosphate buffered saline). After washing 3×, a blocking step wasperformed using 5% non-fat dried milk, followed by titration of humanIgE standards or test supernatants. After washing 3×, biotinylated goatanti-human IgE (Kirkegaard and Perry) was added at a 1:500 dilution.This was followed by further washing and then addition ofstreptavidin-HRP (Zymed) at a 1:500 dilution. After further washing, thereaction was developed using TMB substrate (Kirkegaard and Perry) andabsorbance measured at 520 nm. All washing steps were carried out in PBSplus 0.05% Tween. All incubation steps were performed at volumes of 100μl/well for one hour at room temperature. The sensitivity of this assayis 100 pg/ml.

EXAMPLE 9

This example illustrates the effects of sCD40 and CD40/Fc fusion proteinto inhibit soluble CD23 shedding from IL-4 (5 ng/ml) stimulated B cells.Soluble CD40 and CD40/Fc were tested for their ability to inhibitIL-4-induced sCD23 shedding in a two donor MLC system. The data fromthree experiments are presented in Table 2.

TABLE 2 sCD23 (ng/ml) Exp. 1 Exp. 2 Exp. 3 day day day day day dayAddition 6 12 6 12 6 12 E⁻ + medium 55 <0.5 24 10 10 5 + IL-4 115 55 9662 44 27 + IL-4 + sCD40 (1 μg/ml) nd nd 88 36 38 9 + IL-4 + sCD40 (3μg/ml) 97 4 82 31 40 4 + IL-4 + sCD40 (10 μg/ml) nd nd 72 28 nd nd +IL-4 + IL-7R/Fc (3 μg/ml) 111 48 103 67 40 22 PBM + medium 12 <0.5 15 53 10 + IL-4 39 255 47 22 48 26 + IL-4 + sCD40 (1 μg/ml) nd nd 44 18 4618 + IL-4 + sCD40 (3 μg/ml) 24 6 37 11 45 12 + IL-4 + sCD40 (10 μg/ml)nd nd 28 5 nd nd + IL-4 + IL-7R/Fc (3 μg/ml) 35 26 43 20 50 23

Soluble CD23 levels were measured after 6 and 12 days in culture by acommercial sCD23 ELISA detection kit (Binding Site, San Diego, Calif.).The sensitivity limit was 500 pg/ml. Approximately 1×10⁵ cells per wellwere cultured in triplicate in round-bottomed 96-well microtiter plates(Intermountain Scientific, Bountiful Utah) for the indicated time in thepresence or absence of additives as indicated in Table 2. The resultsshow anti-allergy effects of sCD40. Similar studies were run withCD40/Fc (data not shown) instead of sCD40, and similar results wereobtained. Accordingly, these data in Examples 8 and 9 illustrate ananti-allergy property for CD40.

EXAMPLE 10

This example illustrates B cell proliferative activity of membrane-boundmurine CD40-L for human B cells. Human peripheral blood mononuclearcells (PBMC) were isolated from peripheral blood from normal volunteersby density gradient centrifugation over Histopaque® (Sigma, St. Louis,Mo.) T cell-depleted preparations of cells (E⁻) were obtained byremoving T cells by rosetting with 2-aminoethylisothiouroniumbromide-treated SRBC (sheep red blood cells) and further densitygradient centrifugation over Histopaque®. B cell proliferation assayswere conducted with E⁻ preparations in RPMI media with added 10%heat-inactivated fetal bovine serum (FBS) at 37° C. in a 10% CO₂atmosphere. Approximately 1×10⁵ E⁻ cells per well were cultured intriplicate in flat-bottomed 96-well microtiter plates (Corning) for 7days in the presence of transfected CV1 EBNA cells (described in Example6). The CV1 EBNA cells were transfected with murine CD40-L cDNA or emptyvector. The cells were pulsed with 1 μCi/well of tritiated thymidine (25Ci/nmole Amersham, Arlington Heights, Ill.) for the final eight hours ofculture. Cells were harvested onto glass fiber discs with an automatedcell harvester and incorporated cpm were measured by liquidscintillation spectrometry.

FIG. 4 a shows a comparison of human B cell proliferation of CV1 EBNAcells transfected with empty vector (HAVEO) or with murine CD40-L cDNAin HAVEO vector. These data show that membrane-bound CD40-L stimulateshuman B cell proliferation in the absence of a co-mitogen. FIG. 4 bshows a similar experiment, except that 10 ng/ml of human IL-4 was addedto the cultures. In this experiment, IL-4 slightly enhances the B cellmitogenic activity of membrane-bound murine CD40-L. FIG. 5 is a repeatof the experiment shown in FIG. 4 b. However, when the experiment wasrepeated, there was no evidence of IL-4 co-mitogenic activity. There wasrepeated evidence of CD40-L mitogenic activity. Accordingly,membrane-bound CD40-L stimulates proliferation of human B cells.

EXAMPLE 11

This example illustrates the effect of membrane-bound murine CD40-L tostimulate IgE production and CD23 shedding from E⁻ cells isolated inExample 10. Approximately 1×10⁵ cells/well were cultured in triplicateround bottomed 96-well Nunc microtiter plates (Intermountain Scientific,Bountiful Utah) in Iscove's Modified Dulbecco's Medium (IMDM) plus 10%FCS in a humidified atmosphere of 10% CO₂. Medium was supplemented with50 μg/ml human transferrin (Sigma), 0.5% bovine serum albumin (Sigma)and 1 μg/ml of each of oleic, linoleic and palmitic acids (Sigma). TheE⁻ cells were cultured for 10 days in the presence of 5 ng/ml humanIL-4. A titration of CV1 EBNA cells transfected with murine CD40-L orempty vector were added. After ten days, culture supernatants wereassayed for IgE by the ELISA procedure described in Example 8 or forCD23 shedding by the procedure described in Example 9.

FIG. 6 shows a comparison of IgE production in the supernatants (inng/ml) for cultures of E⁻ cells and CV1 EBNA cells transfected withempty vector (HAVEO) or with CD40-L. No differences were noted with upto 3000 CV1 EBNA cells, however significant IgE production resulted withthe addition of 10000 or 30000 CD40-L transfected CV1 EBNA cells. As acomparison, when E⁻ cells were incubated with medium alone, 5 ng/ml IL-4or 5 ng/ml IL-4 plus 500 ng/ml G28-5 antibody, IgE production was 4.7,2.9 and >600 ng/ml, respectively. When CD23 shedding was measured inFIG. 7, 10000 and 30000 CVI EBNA cells transfected with CD40-L showedincreased CD23 shedding when compared to empty vector control CV1 EBNAcells. As a comparison, when E⁻ cells were incubated with medium alone,5 ng/ml IL-4 or 5 ng/ml IL-4 plus 500 ng/ml G28-5 antibody, CD23shedding was <0.1, 2.4 and 11.2 ng/ml, respectively. These data showthat IgE production and CD23 shedding are both biological activitiesassociated with membrane-bound CD40-L.

EXAMPLE 12

This example illustrates B cell proliferative activity, polyclonalimmunoglobulin (Ig) production, antigen-specific antibody formation andvarious method for using membrane-bound and soluble CD40-L in clinicalapplications. We obtained murine splenic B cells according to proceduresdescribed in Grabstein et al. I supra, Maliszewski et al. I supra andMaliszewski et al. II supra. Briefly, the mixed culture of cells waspurified by T cell depletion using T cell antiserum and complement, andadherent cell depletion by passage of Sephadex® G10 columns and by Bcell positive selection by panning on petri dishes coated with goatanti-mouse IgM. Purified B cells were cultured in RPMI, fetal calf serum(5% for B cell proliferation assays and 20% for plaque forming cellassays or polyclonal antibody assays), 2-mercaptoethanol, antibiotics,amino acids and pyruvate. B cell proliferation was measured according tothe assay described in Example 10 and in Grabstein et al. I supra,Maliszewski et al. I supra and Maliszewski et al. II supra.Antigen-specific antibody formation was measured by the proceduredescribed in Grabstein et al., J. Mol. Cell. Immunol. 2:199, 1986[Grabstein et al. II]. Briefly, antigen specific antibody formation usedsheep red blood cells (SRBC) as antigen (0.03% v/v) in 2.0 ml culturesof 1×10⁶ murine B cells per culture. The B cell cultures were incubatedfor 5 days and plaque forming cells were determined by Jerne hemolyticplaque assay as described in Grabstein et al. II supra. Cell counts weredetermined in a coulter counter. Polyclonal Ig secretion was determinedby isotype-specific ELISA assays in seven day cultures of 1×10⁶ B cellsper 2.0 ml culture as described in Maliszewski et al. I supra andMaliszewski et al. II supra.

The results of B cell proliferation by CV1 EBNA cells transfected withCD40-L or empty vector or 7A1 cells (a T cell helper clone) are shown inFIGS. 8, 10 and 12. These data show that the greatest B cellproliferation was caused by CD40-L. T cell helper cells 7A1 and 7C2 hada minimal effect on B cell proliferation.

The effects of various cells upon antigen specific antibody formationare shown in FIGS. 9 and 11. FIG. 9 shows a comparison of plaque formingcells comparing T cell helper clone 7A1 and murine EL40.9 cells whichsecrete a soluble CD40-L. The EL40.9 cells seem to have an inhibitoryeffect upon antigen specific antibody formation. FIG. 11 shows PFC(plaque forming cells) for T cell helper cells 7C2 and CV1 EBNA cellstransfected with either empty vector or CD40-L. Both 7C2 cells andmembrane-bound CD40-L stimulated antigen specific antibody formation(PFC). FIGS. 13A-13B compare antigen specific antibody formation ofCD40-L and 7A1 cells in the presence or absence of 10 ng/mlinterleukin-2 (IL-2). IL-2 increased PFC for 7A1 cells but did notincrease PFC caused by membrane-bound CD40-L.

Polyclonal Ig production by murine B cells was compared for stimulationor inhibition with membrane-bound CD40-L, control CV1 EBNA cells andhelper T cells 7A1 in the presence of cytokines IL-4 (10 ng/ml) and IL-5(1:40 dilution of COS cell supernatants) or without added cytokines. Theamount of IgA, IgG3, IgE, IgG2b, IgM and IgG1 are shown in Tables 3-8,respectively.

TABLE 3 IgA, ng/ml # CELLS MEDIA +IL-4 + IL-5 CD40-L 2 × 10(5) 666.275 ±174.444  64.639 ± 51.780 1 × 10(5) 288.085 ± 20.773  291.831 ± 10.673 1× 10(4) 53.750 ± 36.531 910.072 ± 62.713 HAVEO 2 × 10(5) 0 628.190 ±42.907 1 × 10(5) 0 477.755 ± 57.478 1 × 10(4) 0 295.640 ± 12.736 7A1(2C11) 1 × 10(6) 0 2177.549 ± 377.052 2 × 10(5) 0 646.898 ± 86.325 1 ×10(5) 0 480.671 ± 40.011 MEDIA 0 458.152 ± 77.258 LPS 88.531 ± 31.248132.336 ± 51.356

TABLE 4 IgG3, ng/ml # CELLS MEDIA +IL-4 + IL-5 CD40-L 2 × 10(5) 108.427± 14.359 0 1 × 10(5) 118.079 ± 8.021  46.535 ± 9.899 1 × 10(4) 127.591 ±6.268  467.023 ± 78.276 HAVEO 2 × 10(5) 0 29.773 ± 5.224 1 × 10(5)11.205 ± 4.434 66.323 ± 8.673 1 × 10(4)  26.389 ± 10.221  34.671 ±12.975 7A1 (2C11) 1 × 10(6) 33.420 ± 9.972 820.856 ± 39.442 2 × 10(5) 0436.074 ± 59.332 1 × 10(5) 0 239.760 ± 45.978 MEDIA 21.808 ± 7.107 64.773 ± 13.924 LPS 816.697 ± 43.553 103.720 ± 11.883

TABLE 5 IgE, ng/ml # CELLS MEDIA +IL-4 + IL-5 CD40-L 2 × 10(5) 0 64.144± 4.979 1 × 10(5) 0 83.493 ± 9.093 1 × 10(4) 0 461.155 ± 60.514 HAVEO 2× 10(5) 0 0 1 × 10(5) 0 4.208 ± .527 1 × 10(4) 0 0 7A1 (2C11) 1 × 10(6)0 208.091 ± 8.090  2 × 10(5) 0 32.530 ± 0.723 1 × 10(5) 0 15.889 ± 2.947MEDIA 0 12.602 ± 1.460 LPS 0 408.355 ± 9.764 

TABLE 6 IgG2b, ng/ml # CELLS MEDIA +IL-4 + IL-5 CD40-L 2 × 10(5) 0 0 1 ×10(5) 0 6.230 ± .285  1 × 10(4) 0 47.414 ± .241  HAVEO 2 × 10(5) 0 7.001± 2.358 1 × 10(5) 0 6.230 ± 2.285 1 × 10(4) 0 9.620 ± 2.650 7A1 (2C11) 1× 10(6) 0 189.343 ± 2.837  2 × 10(5) 0 22.431 ± 6.835  1 × 10(5) 0 7.207± 1.580 MEDIA 0 7.422 ± 1.602 LPS 0 33.291 ± 3.183 

TABLE 7 IgM, μg/ml # CELLS MEDIA +IL-4 + IL-5 CD40-L 2 × 10(5) 1.805 ±0.639  0.439 ± 0.184 1 × 10(5) 2.237 ± 0.583  5.878 ± 0.858 1 × 10(4)2.293 ± 0.595  96.730 ± 13.009 HAVEO 2 × 10(5) 0 10.890 ± 2.126 1 ×10(5) 0 13.303 ± 0.993 1 × 10(4) 0.624 ± 0.178 22.538 ± 2.304 7A1 (2C11)1 × 10(6) 0.769 ± 0.124 104.857 ± 17.463 2 × 10(5) 0.142 ± 0.052 27.016± 1.706 1 × 10(5) 0.126 ± 0.048 13.070 ± 0.600 MEDIA 0.231 ± 0.05736.809 ± 2.860 LPS 53.302 ± 9.668  41.974 ± 6.158

TABLE 8 IgG1, ng/ml # CELLS MEDIA +IL-4 + IL-5 CD40-L 2 × 10(5) 0130.185 ± 24.547 1 × 10(5) 0 310.588 ± 1.261  1 × 10(4) 0 270.727 ±17.511 HAVEO 2 × 10(5) 0 187.668 ± 57.730 1 × 10(5) 0  43.320 ± 49.770 1× 10(4) 0 1363.464 ± 45.841  7A1 (2C11) 1 × 10(6) 0  145.652 ± 136.070 2× 10(5) 0 365.563 ± 24.276 1 × 10(5) 0  449.475 ± 101.012 MEDIA 0 133.660 ± 386.231 LPS 0 246.213 ± 21.526

These data indicate that the interaction of CD40 with its ligand is theprincipal molecular interaction responsible for T cell contact dependentinduction of B cell growth and differentiation to both antigen-specificantibody production and polyclonal Ig secretion. As such, these datasuggest that antagonists of this interaction, by soluble CD40, CD40/Fcfusion protein and possibly soluble CD40-L (monomeric), willsignificantly interfere with development of antibody responses.Therefore clinical situations where CD40, CD40/Fc fusion proteins andsoluble CD40-L include allergy, lupus, rheumatoid arthritis, insulindependent diabetes mellitus, and any other diseases where autoimmuneantibody or antigen/antibody complexes are responsible for clinicalpathology of the disease. Moreover, membrane-bound CD40-L or oligomericsoluble CD40-L will be useful to stimulate B cell proliferation andantibody production. As such, these forms of CD40-L are most useful forvaccine adjuvants and as a stimulating agent for mAb secretion fromhybridoma cells.

EXAMPLE 13

This example illustrates the effect of membrane-bound CD40-L uponproliferation of and IgE secretion from peripheral blood mononuclearcells (E⁻). E⁻ cells were obtained according to the procedure describedin Example 10 and incubated for 7 or 10 days in the presence of CV1 EBNAcells transfected with empty vector or mCD40-L cDNA. Additionally,CD40/Fc fusion protein (described in Example 1) or TNF Receptor/Fcfusion protein (described in WO 91/03553) was added to some of thepreparations as indicated in FIGS. 14A-14B. IgE secretion was measuredaccording to the procedure described in Example 8 and B cellproliferation was measured according to the procedure described inExample 10.

The results for B cell proliferation and IgE secretion are shown inFIGS. 14A-14B for five different concentrations of transfected CV1 EBNAcells. Both B cell proliferation and IgE secretion were increased in thepresence of membrane-bound CD40-L. Addition of CD40/Fc fusion proteinablated both B cell proliferation and IgE secretion. The TNF Receptor/Fcfusion protein had no effect. As a comparison for IgE secretion,addition of IL-4 as a control agent (without transfected CV1 EBNA cells)produced no IgE in this assay and addition of IL-4 plus G28-5 anti-CD40mAb resulted in 29.7 ng/ml IgE in this assay.

EXAMPLE 14

This example describes construction of a CD40-L/Fc DNA construct toexpress a soluble CD40-L/Fc fusion protein referred to as CD40-L/FC2construct. DNA encoding CD40-L/FC2 comprises sequences encoding a leader(or signal) peptide, an eight amino acid hydrophilic sequence describedby Hopp et al. (Hopp et al., Bio/Technology 6:1204, 1988; referred to asFlag®), a suitable Fc region of an immunoglobulin, a [Gly₄Ser]₃ repeatsequence (described in U.S. Pat. No. 5,073,627, which is incorporated byreference herein) or other suitable linker sequence, and theextracellular region of human CD40-L from amino acid 51 to amino acid261 (SEQ ID NO:11). A pDC406 expression vector containing a leadersequence, Flag®, and human IgG₁ Fc is prepared using conventionaltechniques of enzyme cutting and ligation of fragments encoding a leadersequence, Flag®, and human IgG₁ Fc, and restricted with Nsi 1 and Not 1.

A PCR technique (Saiki et al., Science 239:487, 1988) was employed using5′ (upstream) and 3′ (downstream) oligonucleotide primers to amplify theDNA sequences encoding CD40 extracellular ligand binding domain from acloning vector containing human CD40-L (ATCC 68873; SEQ ID NO: 11) toform a PCR fragment. The upstream oligonucleotide primer (SEQ ID NO:13)introduced a Nsi 1 site upstream from a linker sequence([Gly₄Ser]₃SerSer), which was followed by 21 nucleotides of theextracellular domain of CD40-L (amino acids 51 through 57 of SEQ ID NO:11). A downstream oligonucleotide primer (SEQ ID NO:14) introduced a Not1 site just downstream of the termination codon of the CD40-L. The PCRfragment was then ligated into the pDC406 expression vector containing aleader sequence, Flag®, and human IgG₁ Fc. The nucleotide and predictedamino acid sequence of CD40-L/FC2 are presented in SEQ ID NO:15 and SEQID NO:16. The resultant DNA construct (CD40-L/FC2) was transfected intothe monkey kidney cell line CV-1/EBNA (ATCC CRL 10478). The constructencoded a soluble CD40-L capable of binding CD40, as evidenced bybinding observed in fluorescence-activated cell sorting (FACS) analysisusing cells that express CD40.

Large scale cultures of human embryonic kidney 293 cells (ATCC CRL 1573)transfected with the construct encoding CD40-L/FC2 were grown toaccumulate supernatant containing CD40-L/FC2. The 293 cell line, apermanent line of primary human embryonal kidney transformed by humanadenovirus 5 DNA, permits expression of recombinant proteins ligatedinto the pCD406 vector. The CD40-L/FC2 fusion protein in supernatantfluid was purified by affinity purification. Briefly, culturesupernatant containing the CD40-L/FC2 fusion protein was purified byfiltering mammalian cell supernatants (e.g., in a 0.45μ filter) andapplying filtrate to an antibody affinity column comprising biotinylatedgoat anti-human IgG (Jackson Immunoresearch Laboratories, Inc.,Westgrove, Pa., USA) coupled to Streptavidin-agarose (Pierce Chemical,Rockford, Ill., USA) at 4° C., at a flow rate of approximately 60 to 80ml/hr for a 1.5 cm×12.0 cm column. The column was washed withapproximately 20 column volumes of PBS (phosphate buffered saline),until free protein could not be detected in wash buffer. Bound fusionprotein was eluted from the column with 12.5 mM citrate buffer, 75 mMNaCl, pH 2.8, and brought to pH 7 with 500 mM Hepes buffer, pH 9.1. Thepurified, oligomeric CD40-L/FC2 peptide induced human B cellproliferation in the absence of any co-stimuli, and (in conjunction withthe appropriate cytokine) resulted in the production of IgG, IgE, IgAand IgM, as described in Example 12 for membrane-bound CD40-L.

EXAMPLE 15

This example describes construction of a CD40-L DNA construct to expressa soluble CD40-L fusion protein referred to as trimeric CD40-L. TrimericCD40-L contains a leader sequence, a 33 amino acid sequence referred toas a “leucine zipper” (SEQ ID NO:17), and an eight amino acidhydrophilic sequence described by Hopp et al. (Hopp et al.,Bio/Technology 6:1204, 1988; referred to as Flag®), followed by theextracellular region of human CD40-L from amino acid 51 to amino acid261 (SEQ ID NO:11). The utility of the leader and the Flag® sequenceshave been described in the Detailed Description. The 33 amino acidsequence presented in SEQ ID NO:17 trimerizes spontaneously in solution.Fusion proteins comprising this 33 amino acid sequence are thus expectedto form trimers or multimers spontaneously.

The construct is prepared by synthesizing oligonucleotides representinga leader sequence, the 33 amino acid sequence described above, and theFlag® sequence, then ligating the final product to a DNA fragmentencoding amino acids 51 through 261 of SEQ ID NO:11, prepared asdescribed in Example 14.

The resulting ligation product in expression vector pDC406 wastransfected into the monkey kidney cell line CV-1/EBNA (ATCC CRL 10478).The pDC406 plasmid includes regulatory sequences derived from CD40,human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV). TheCV-1/EBNA cell line was derived by transfection of the CV-1 cell linewith a gene encoding Epstein-Barr virus nuclear antigen-1 (EBNA-1) thatconstitutively expresses EBNA-1 driven from the human CMVintermediate-early enhancer/promoter. The EBNA-1 gene allows forepisomal replication of expression vectors, such as pDC406, that containthe EBV origin of replication.

Once cells expressing the fusion construct are identified, large scalecultures of transfected cells are grown to accumulate supernatant fromcells expressing trimeric CD40-L. The trimeric CD40-L fusion protein insupernatant fluid is purified by affinity purification substantially asdescribed in U.S. Pat. No. 5,011,912. Silver-stained SDS gels of theeluted CD40-L fusion protein can be prepared to determine purity.

EXAMPLE 16

This example describes two solid-phase binding assays, the first ofwhich, (a), can be used to asses the ability of trimeric CD40-L to bindCD40, and the second of which, (b), is used to detect the presence ofCD40-L.

(a) Quantitative CD40-L ELISA

CD40/Fc is prepared and purified as described Example 1, and used tocoat 96-well plates (Corning EasyWash ELISA plates, Corning, NY, USA).The plates are coated with 2.5 μg/ml well of CD40/Fc in PBS overnight at4° C., and blocked with 1% non-fat milk in PBS for 1 hour at roomtemperature. Samples to be tested are diluted in 10% normal goat serumin PBS, and 50 μl is added per well. A titration of unknown samples isrun in duplicate, and a titration of reference standard of CD40-L is runto generate a standard curve. The plates are incubated with the samplesand controls for 45 minutes at room temperature, then washed four timeswith PBS. Second step reagent, rabbit anti leucine zipper, is added (50μl/well, concentration approximately 2.5 μg/ml), and the plates areincubated at room temperature for 45 minutes. The plates are againwashed as previously described, and goat F(ab′)2 anti-rabbit IgGconjugated to horseradish peroxidase (Tago, Burlingame, Calif., USA) isadded. Plates are incubated for 45 minutes at room temperature, washedas described, and the presence of CD40-L is detected by the addition ofchromogen, tetramethyl benzidene (TMB; 100 μl/well) for 15 minutes atroom temperature. The chromogenic reaction is stopped by the addition of100 μl/well 2N H₂SO₄, and the OD₄₅₀-OD₅₆₂ of the well determined. Thequantity of trimeric CD40-L can be determined by comparing the OD valuesobtained with the unknown samples to the values generated for thestandard curve. Values are expressed as the number of binding units perml. A binding unit is roughly one ng of protein as estimated using apurified Fc fusion protein of the ligand as a standard. In this manner,the concentration and specific activity of several different batches oftrimeric CD40-L purified as described in Example 19 have beendetermined.

(b) Qualitative Dot Blot

CD40-L trimer (1 μl of crude supernatant or column fractions) isadsorbed to dry BA85/21 nitrocellulose membranes (Schleicher andSchuell, Keene, N.H.) and allowed to dry. The membranes are incubated intissue culture dishes for one hour in Tris (0.05 M) buffered saline(0.15 M) pH 7.5 containing 1% w/v BSA to block nonspecific bindingsites. At the end of this time, the membranes are washed three times inPBS, and rabbit anti-leucine zipper antibody is added at an approximateconcentration of 10 μg/ml in PBS containing 1% BSA, following which themembranes are incubated for one hour at room temperature. The membranesare again washed as described, and a horseradish peroxidase(HRP)-labeled antibody (such as goat anti-rabbit Ig; Southern Biotech,Birmingham, Ala.) at an approximate dilution of 1:1000 in PBS containing1% BSA is added. After incubating for one hour at room temperature, themembranes are washed and chromogen (i.e. 4-chloronaphthol reagent,Kirkegard and Perry, Gaithersburg, Md.) is added. Color is allowed todevelop for ten minutes at room temperature, and the reaction is stoppedby rinsing the membranes with water. The membranes are washed, and thepresence of CD40-L is determined by analyzing for the presence of ablue-black color. This assay was used to determine the presence orabsence of trimeric CD40-L in cell culture supernatant fluids and inpurification column fractions. The assay further provides asemi-quantitative method of determining relative amounts of trimericCD40-L by comparing the intensity of the color in unknown samples to theintensity of known quantities of controls.

EXAMPLE 17

This example describes construction of a human CD40-L DNA construct toexpress trimeric CD40-L in Chinese hamster ovary (CHO) cells. Asdescribed in Example 15, trimeric CD40-L contains a leader sequence, anda 33 amino acid sequence referred to as a “leucine zipper” (SEQ IDNO:17), followed by the extracellular region of human CD40-L from aminoacid 51 to amino acid 261 (SEQ ID NO:11). The construct was prepared bycutting the appropriate DNA from the a plasmid containing human CD40-L(derived from the plasmid described in Example 15), and ligating the DNAinto the expression vector pCAVDHFR. The resultant construct wasreferred to as CAV/DHFR-CD40LT. pCAVDHFR includes regulatory sequencesderived from cytomegalovirus, CD40, and Adenovirus 2, along with thegene for dihydrofolate reductase (DHFR), and allows random integrationof a desired gene into host cell chromosomes. Expression of DHFR enablesthe DHFR⁻ host cells to grow in media lacking glycine, hypoxanthine, andthymidine (GHT). A similar construct was also made for expression ofmurine CD40-L timer in CHO cells. In addition to the leader and leucinezipper sequences, the murine construct also contained a sequenceencoding the octapeptide referred to as Flag® (described previously)between the trimerization domain (leucine zipper) and the extracellularregion of murine CD40-L. The nucleotide and amino acid sequence of thehuman and murine trimeric CD40-L-encoding DNAs are shown in SEQ ID NOs20 and 22 respectively. Additional constructs can be prepared usingstandard methods. For example, vectors which incorporates dual promoterssuch as those described in U.S. Pat. No. 4,656,134, or vectors employingenhancer sequences such as those described in U.S. Pat. No. 4,937,190 orin Kaufman et al., Nucl. Acids Res. 19:4485, 1991, are also useful inpreparing constructs for expression of CD40-L in CHO cells.

The resulting ligation product was transfected into CHO cells usingeither Lipofectin® Reagent or Lipofectamine™ Reagent (Gibco BRL,Gaithersburg, Md.). Both of these reagents are commercially availablereagents used to form lipid-nucleic acid complexes (or liposomes) which,when applied to cultured cells, facilitate uptake of the nucleic acidinto the cells. Cells which were transfected with the pCAVDHFR-CD40LTconstruct were selected in DMEM:F12 medium in the absence of GHT. Cellswhich were able to grow in the absence of GHT were tested for productionof CD40-L using a solid phase binding assay as described in Example 16.Results indicted that in this transfection system, Lipofectamine™Reagent gave higher rates of successful transfection.

Approximately 160 clones were screened and two positive clones wereidentified and expanded for further study. Cells were passaged inGHT-free DMEM:F12 medium, and monitored for stability for assessingproduction of trimeric CD40-L in the solid-phase binding assay describedabove. Based on these results, one clone was chosen which appeared to bestabley transfected with the CD40-L DNA, and which produced and secretedapproximately 1 μg/10⁶ cells/day of CD40-L trimer. Additional constructscomprising other vectors and all or a portion of the DNA sequencesdescribed in this example can be used to prepare additional stablytransfected cell lines, substantially as described herein. For example,constructs encoding monomeric CD40-L similar to those described inExample 18 can be prepared, as can plasmids encoding any of thepreviously described constructs.

Once such stably transfected cells were identified, large scale culturesof transfected cells were grown to accumulate supernatant containingtrimeric CD40-L. Suitable large-scale culture conditions include the useof bioreactors, as described below in Example 19. Similar procedureswere followed to produce CHO cell lines that secreted a trimeric murineCD40-L at approximately 0.05 μg/10⁶ cells/day. CHO cells stablytransfected with either the human or murine CD40-L construct, havingacquired a DHFR gene from the pCAVDHFR plasmid, are resistant tomethotrexate. Methotrexate can be added to the culture medium to amplifythe number of copies of the CD40-L trimer DNA in order to increaseproduction of CD40-L trimer.

EXAMPLE 18

This example describes construction of a murine CD40-L DNA construct toexpress a soluble CD40-L protein referred to as monomeric CD40-L.Monomeric CD40-L contains a leader sequence, an eight amino acidsequence referred to as Flag® (amino acids 1-8 of SEQ ID NO:16),followed by the amino terminal truncated region of CD40-L encompassingthe extracellular B-sheet forming region of the CD40 molecule from aminoacid 119 to 260 of SEQ ID NO:1. A 58 amino acid stretch of theextracellular spacer region of the CD40-L molecule (amino acids 51-118of SEQ ID NO:1) has been deleted in this construct, as has thetransmembrane region (amino acids 1-50 of SEQ ID NO:1).

A PCR technique using 5′ (upstream) and 3′ (downstream) oligonucleotideprimers was used to amplify the DNA sequences encoding the CD40-Ltruncated extracellular domain from a cloning vector containing murineCD40-L. The upstream oligonucleotide primer(ATATGAATTCGACTACAAAGATGACGATGATAAACCTCAAATTGCAGCACACGTT; SEQ ID NO:18)appended an EcoRI site and the Flag coding sequence upstream from CD40nucleotide 355. The downstream oligonucleotide primer(CCTTCGCGGCCGCGTTCAGAGTTT GAGTAAGCCAA, SEQ ID NO:19) introduced a Not 1site downstream of the authentic termination codon of the CD40L.

The PCR fragment was ligated into the multiple cloning site(EcroRI/NotI) of the baculovirus expression vector pAcGP67A (PharMingen,San Diego, Calif.) which contains the signal sequence for a glycoproteinof the Autographica californica nuclear polyhedrosis virus under thecontrol of the viral polyhedrin promoter. The resultant DNA constructwas cotransfected with Autographica californica viral DNA intoSpodoptera frugiperda cells (SF21), and the resultant recombinant viruswas plaque purified.

The CD40-L protein encoded by this construct was purified by Flag®affinity chromatography from serum free culture of recombinant virusinfected cells. Purified protein had an apparent molecular weight of 21Kd when run in a reducing PAGE and stained with Coomasie Blue. Bothcrude infected cell supernatants containing CD40-L and affinity purifiedCD40-L protein showed receptor binding activity in a solid phase assayutilizing the CD40Fc recombinant receptor. Mock infected controls had noactivity.

A similar CD40-L construct was made without an amino terminal Flag®sequence. This construct utilized an existing Bam HI site at nucleotide351 in the CD40-L sequence and the downstream PCR oligonucleotide primerdescribed above (SEQ ID NO:19). After amplification of the CD40 sequencewith a 5′ upstream oligonucleotide homologous to CD40-L nucleotides324-346, and the downstream primer which introduced a Not I site, thePCR product was cut with Bam HI and Not I and ligated into pAcGP67A cutwith Bam HI and NotI. This construct was cotransfected into SF21 cellsalong with viral DNA as previously described, and recombinant virus wasplaque purified, expanded and used to infect insect cells to produceserum free conditioned supernatants. CD40-L was detectable in thesecrude supernatants by both CD40Fc receptor binding assay and bydetection of an 18 Kd band on a Coomassie Blue-stained PAGE.

EXAMPLE 19

This example describes purification of trimeric murine CD40L fromsupernatant fluid from transfected CHO cells. A CHO cell line expressingmuCD40LT was maintained in suspension in spinner-flask cultures. Forproduction, the cells were centrifuged and resuspended into a controlled3 liter bioreactor in serum-free medium. Oxygen, agitation and pH werecontrolled for at 40% dissolved O₂ (relative to air saturation), 150 RPMand 7.2, respectively. The culture was harvested after nine days. Thetotal volume of approximately 160 ml of supernatant fluid from thebioreactor was dialyzed overnight at 4° C. against 4 L of 20 mM Tris pH7.5 buffer containing 150 mM NaCl, and then adjusted to 1M (NH₄)₂SO₄ bythe addition of solid (NH₄)₂SO₄. Dialysis accomplished the removal oflow-molecular weight contaminants; other techniques will also be usefulfor this purpose, for example, constant volume diafiltration.

The dialyzed supernatant was initially purified by hydrophobicinteraction chromatography. The supernatant was applied to a 1.6×13 cm(26 ml) Phenyl Sepharose® CL-4B column (Pharmacia, Uppsala, Sweden)previously equlibrated with 10 mM Tris pH 8.0/1M (NH₄)₂SO₄ (Buffer A).The column was washed with 60 mL Buffer A, and bound proteins wereeluted at 2 mL/min with a decreasing (NH₄)₂SO₄ gradient using Buffer Aand 10 mM Tris pH 8.0 (Buffer B). The gradient conditions were 0 to 60%Buffer B in 20 ml, hold at 60% Buffer B for 60 ml, 60 to 100% Buffer Bin 20 ml, and hold at 100% Buffer B for 60 ml. A total of 53 3 mlfractions were collected during the elution process. The elution ofprotein was monitored by absorbance at 280 nm. The presence of activetrimeric CD40L was determined by an ELISA as described in Example 16. Apeak of activity eluted in fractions 8-20. In a subsequent purificationrun, highsub and lowsub Phenyl Sepharose® 6 Fast Flow (Pharmacia,Uppsala, Sweden) were used for the hydrophobic interaction step; thehighsub Phenyl Sepharose® column was found to give equivalent results tothose obtained with Phenyl Sepharose® CL-4B.

Peak fractions from the Phenyl Sepharose® CL-4B column were pooled, andglycerol was added to a final concentration of 10% (v/v). The pool wasthen concentrated to a volume of approximately 4.5 ml using AmiconCentriprep 10 concentrators with a 10,000 molecular weight cutoff, andchromatographed over a sizing column (Superdex 200 26/60; Pharmacia,Uppsala, Sweden; 2.6×60 cm). The concentrated pool was loaded, andeluted with 20 mM Tris pH 7.5/150 mM NaCl/10% glycerol (v/v), at a flowrate of 2.0 ml/min. Protein elution was monitored at 280 nm, and eighty2 ml fractions were collected. Activity was determined as describedabove; a peak of activity eluted in fractions 36-48. Purity wasevaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) under reducing conditions on 10% acrylamide gels (Novex). Thegels were stained by silver stain substantially as described by Oakleyet al., Anal. Biochem. 105:361 (1980). The silver-stained gels showedseveral proteins present in the fractions.

Fractions from the peak of activity from the Superdex 200 column werepooled, concentrated as described above to approximately 2.0 ml, diluted1:2 in 20 mM Bis Tris Propane pH6.5/10% glycerol (v/v), and furtherpurified by anion exchange chromatography. The concentrated pooledmaterial was applied at 1 mL/min to a Mono Q column (Pharmacia, 0.5×5cm) equlibrated with 20 mM Bis Tris Propane pH6.5/10% glycerol (v/v)(Buffer A). The column was washed with 16 mL Buffer A and eluted with asalt gradient using Buffer A and 20 mM Bis Tris Propane pH6.5/500 mMNaCl/10% glycerol (v/v) (Buffer B). The column elution conditions were 0to 60% Buffer B in 20 ml, 60 to 100% Buffer B in 1.0 ml, and hold at100% Buffer B for 10 ml. A total of 30 1 ml fractions were collectedduring the elution process. Activity and A₂₈₀ were monitored asdescribed previously. A peak of activity eluted in fractions 15-23. Thefractions were evaluated by SDS-PAGE and silver stain as describedabove. Fractions 20-22 were estimated to contain about 80% trimericmurine CD40L, and were pooled. In a subsequent run, a High-performance Qresin (Pharmacia, Uppsala, Sweden) was used and found to give equivalentresults. Table 9 below summarizes the results of the procedure used topurify trimeric murine CD40L.

TABLE 9 Purification of Trimeric Murine CD40-L Total # Total VolumeBinding Protein Specific Step (ml) Units (mg) Activity* 1. Supernatantfluid 160 5.2 × 10⁶ 280 1.9 × 10⁴ 2. Dialyzed Supernatant 169 6.4 × 10⁶106 6.0 × 10⁴ 3. Phenyl Sepharose ® 37 2.3 × 10⁶ 8.0 2.9 × 10⁵ pool 4.Superdex 200 pool 24 2.3 × 10⁶ 1.4 1.6 × 10⁵ 5. Mono Q pool 7 6.4 × 10⁵0.91 7.0 × 10⁵ *Specific activity is defined as the number of bindingunits of CD40-L per mg protein. One binding unit of CD40L is defined as0.5 ng of purified CD40-L, as determined in a quantitative, enzyme-basedbinding assay. Protein concentration was determined using the BCAProtein Assay Reagent (Pierce); bovine serum albumen was used as thestandard.

EXAMPLE 20

This example describes the effect of CD40-L trimer (CD40LT) on primaryantibody response to a T-dependent antigen. On day 0, 6 BALB/c mice wereinjected subcutaneously with 200 μl of a suspension containing 10 μg ofovalbumen (OVA), in the presence of Freund's incomplete adjuvant (IFA).Three of the mice also received 200 μl of PBS containing a total of 1.5μg CD40LT, while the remaining mice received a similar amount of acontrol protein (murine serum immunoglobulin; msIgG). The mice wereagain treated with 1.5 μg of CD40LT or control protein on day 6.

Serum samples were taken on days 7 and 14, and analyzed for elevatedlevels of antigen-specific IgG or IgM using an OVA ELISA. Briefly,96-well plates were coated with 10 μg/well of OVA at 4° C. overnight,then blocked with non-fat milk. Serial two-fold dilutions of serumsamples were prepared in PBS containing 10% normal goat serum, and 50 μlof each dilution was added to a well. Plates were incubated for one hourat room temperature, and washed with PBS. The presence ofantigen-specific IgG or IgM was detected using goat anti mouse IgG orIgM (Southern Biotech) conjugated to horseradish peroxidase for one hourat room temperature, followed by a wash step and the addition ofsubstrate (TMB, Kirkegard and Perry). Color development proceeded forten minutes at room temperature, and was stopped by the addition ofH₂SO₄. The maximal dilution of serum dilution containing IgG or IgManti-OVA activity was determined by plotting the OD₄₅₀-OD₅₆₂ of thediluted mouse sera, and comparing the OD values obtained with OD valuesfrom pre-immune sera. Results are presented in Table 10 below.

TABLE 10 Effect of Trimeric Murine CD40-L on Primary Immune ResponseTotal IgG^(a) Relative Ab levels^(b) Endpoint titer (est^(c)) % overnon-immune_(max) Treatment: Day 7 Day 14 Day 7 Day 14 msIgG (#1) <50 400<25 216 msIgG (#2) <50 >400 <25 285 msIgG (#3) <50 200 <25 212 CD40LT(#1) 400 200 279 240 CD40LT (#2) 800 >400 379 339 CD40LT (#3) 200 200178 228 ^(a)Antigen-specific IgM titers were low at both day 7 and day14 in both groups. ^(b)(OD_(max) of treated mouse/OD_(max) of pre-immunecontrol) × 100 ^(c)Estimated.

The mice treated with CD40LT exhibited greater levels of OVA-specificIgG as compared to control mice, indicating that CD40LT was able toboost a primary immune response to a T-dependent antigen, both enhancingthe level of antigen-specific antibody and isotype switching from IgM toIgG.

A second experiment was carried out using different lots of reagents andvarying the concentrations of the CD40-L. A significant differencebetween the control mice and the mice treated with CD40 ligand was notobserved at day 7, however, CD40-L did enhance the day 14 response.Additional experiments to address the use of CD40-L will include ananalysis of different antigens as well as the use of different adjuvantsand delivery systems.

EXAMPLE 21

This example illustrates the activities of monoclonal antibodies toCD40-L. Supernatants from 264 hybridomas prepared as described inExample 7 were screened for anti-CD40-L activity by FACS analysis usinghuman peripheral blood T cells stimulated with PMA and ionomycin for 16hours. Under these conditions, four of the tested hybridoma supernatantsgave a FACS profile similar to that obtained with CD40/Fc; an exemplaryFACS profile is shown in FIGS. 15A-15D. Six additional hybridomasupernatants gave weak positive results, and the remainder did notappear to bind activated T cells.

The ten hybridoma supernatants that gave positive or weak positiveresults were then tested in another FACS assay using CV-1/EBNA cellstransfected with vector alone or with vector encoding human CD40-L, aswell as being reevaluated against activated T cells. Several of thesupernatants appeared to be non-specifically reactive, however, threesupernatants specifically stained CD40-L expressing CV-1/EBNA cells, andwere selected for cloning and further evaluation.

The three anti-CD40-L secreting clones were expanded and supernatantfluids were evaluated for ability to bind CD40-L and inhibit (or block)binding of CD40-L to CD40. In a FACS assay using activated human T cells(prepared as described above), the monoclonal antibody secreted by threeof the clones blocked the binding of CD40/Fc to CD40-L, whereas thefourth did not. Representative results are shown in FIGS. 16A-16D.Several of the monoclonal antibodies were also tested for the ability toinhibit B cell proliferation in an assay substantially as described inExample 13 herein, using CD40-L-containing supernatant fluid from COScells transfected with a vector encoding CD40-L. As shown in FIG. 17,the monoclonal antibodies that were able to inhibit binding of CD40/Fcto CD40-L by FACS analysis also inhibited the ability of trimeric CD40-Lplus anti-IgM to induce proliferation of peripheral blood B cells.

The monoclonal antibodies were also evaluated in a solid phase ELISA inwhich plates were coated with a rabbit antibody to the leucine zipperdomain of trimeric CD40-L. Trimeric CD40-L was then added to the plates,followed by (after appropriate incubation and washing steps) supernatantfluids containing the monoclonal antibodies. The presence of antibodiesto the CD40-L trimer was detected using enzyme labeled anti-mouseimmunoglobulin followed by the appropriate substrate. The threemonoclonal antibodies that inhibited the binding of CD40/c to CD40-Lexpressing T cells by FACS also bound to the CD40-L trimer used in thesolid phase ELISA. Two of the hybridoma cell lines (designatedhCD40L-M90 and hCD40L-M91) were selected for further expansion. Thesehybridoma cell have been deposited with the American Type CultureCollection 12301 Parklawn Drive, Rockville, Md. 20852, USA under termsof the Budapest treaty on Feb. 23, 1996, and given accession numberHB-1205 (hCD40L-M90) and HB-12056 (hCD40L-M91).

EXAMPLE 22

This example illustrates the binding affinities of several differentCD40-L constructs. Affinity experiments were conducted by biospecificinteraction analysis (BIA) using a biosensor, an instrument thatcombines a biological recognition mechanism with a sensing device ortransducer. An exemplary biosensor is BIAcore™, from Pharmacia BiosensorAB (Uppsala, Sweden; see Fägerstam L. G., Techniques in ProteinChemistry II, ed. J. J. Villafranca, Acad. Press, NY, 1991). BIAcore™uses the optical phenomenon surface plasmon resonance (Kretschmann andRaether, Z. Naturforschung, Teil. A 23:2135, 1968) to monitor theinteraction of two biological molecules. Molecule pairs having affinityconstants in the range 10⁵ to 10¹⁰ M⁻¹, and association rate constantsin the range of 10³ to 10⁶ M⁻¹s⁻¹, are suitable for characterizationwith BIAcore™.

The biosensor chips were coated with goat anti-human IgG₁ Fc, which wasused to bind CD40/Fc (prepared as described in Example 1) to the chip.The different constructs of CD40-L were then added at increasingconcentrations; the chip was regenerated between the differentconstructs by the addition of sodium hydroxide. Two separate experimentswere performed. In the first, the binding of a dimeric human CD40-L(Example 14), trimeric human CD40-L (Example 15), dimeric murine CD40-L(prepared substantially as described in Example 14 for human CDr0-L/FC)and trimeric murine CD40-L (prepared substantially as described for thehuman CD40-L in Example 15) were compared. In the second experiment, thebinding of trimeric human CD40-L was compared to the binding of twodifferent preparations of monomeric human CD40-L prepared as describedin Example 18. The resultant data were analyzed to determine theaffinity and association rate constants of the different CD40-Lconstructs. Results are shown in Table 12 below, and in FIGS. 18 and19A-19B.

TABLE 12 Binding of CD40-L to CD40/Fc Site 1 Site 2 (mol/mol K₁ (mol/molK₂ CD40/Fc) (M⁻¹) CD40/Fc) (M⁻¹) Human 0.04 ± 0.02 5 ± 3 × 10⁹  0.68 ±0.07 7.5 ± 2.5 × 10⁷ Trimer Human 0.013 ± 0.002 1.6 ± 0.7 × 0.049 ±0.004 7.0 ± 2.1 × 10⁸ Dimer 10¹¹ Murine  0.02 ± 0.003 6 ± 3 × 10¹⁰ 0.44± 0.02 1.6 ± 0.2 × 10⁸ Trimer Murine 0.05 ± 0.02 7 ± 4 × 10⁹  0.14 ±0.23 4.0 ± 1.0 × 10⁷ Dimer Human 0.26 6.6 × 10⁸ 0.41 2.6 × 10⁷ TrimerMono- Not Detected Not Detected 1.20 4.6 × 10⁷ mer #1 Mono- Not DetectedNot Detected 1.27 1.1 × 10⁷ mer #2

Analysis of the data indicated that a CD40-L monomer comprising solelythe portion of the extracellular domain most homologous to TNF wascapable of binding CD40, although with somewhat lower affinity thanoligomeric CD40-L. An analysis of the ratio of binding in the secondexperiment demonstrated that there are twice as many CD40-L monomerunits bound per CD40/Fc molecule as trimeric CD40-L, confirming that twomonomers of CD40-L bind one CD40/Fc dimer and one trimeric CD40-L bindsone CD40/Fc dimer.

EXAMPLE 23

This example demonstrates that CD40-L enhances the generation ofcytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). A4-hour ⁵¹Cr release assay was used to assess the cytolytic activity ofhuman T cells essentially as described in Alderson et al., J. Exp. Med.172:577 (1990). Briefly, freshly isolated peripheral blood mononuclearcells from one donor were cultured in MLC (mixed lymphocyte culture)with irradiated, allogeneic stimulating cells (target cells), either inthe presence or absence of membrane-bound CD40-L. ⁵¹Cr-labeled targetcells were prepared by incubating tumor cell lines, or three day PHAblasts from a second donor, with 100 μCi of ⁵¹Cr for one hour at 37° C.

Cell cultures to be assessed for cytolytic activity were washed twice inculture medium and serially diluted in 96-well V-bottom plates (Costar).⁵¹Cr-labeled target cells (2×10³) were added to each well (total volumeof 200 μl/well), and the plates were incubated for four hours at 37° C.After incubation, the plates were centrifuged at 150 g for five minutes,and harvested using a Skatron SCS harvesting system (Skatron, Sterling,Va.). ⁵¹Cr content of the supernatants was determined using a MicromedicME Plus gamma scintillation counter (Micromedic, Huntsville, Tenn.).Percent specific ⁵¹Cr release was calculated according to the formula100×(experimental cpm−spontaneous cpm)/(maximum cpm/spontaneous cpm)where spontaneous cpm=cpm released in the absence of effector cells andmaximum cpm=cpm released in the presence of 1N HCl. The results of thisexperiment indicated that membrane-bound CD40-L enhanced CTL generation.A polyclonal anti-IL-2 antiserum capable of neutralizing 10 ng/ml ofIL-2 at a 1:500 dilution was used to demonstrate that CD40-L enhancementof CTL had both IL-2 dependent and IL-2 independent components.

Similar experiments were performed to analyze the phenotype of theresponding cells. T cells were purified by rosetting with2-aminoethylisothiouronium bromide hydrobromide-treated sheep red bloodcells. CD4+ and CD8+ populations were further purified usingimmunomagnetic selection using a MACS (Milenyi Biotec, Sunnyvale,Calif.) according to the manufacturer's protocol. Whereas IL-2 enhancedCTL generation by PBMC, purified T cells and CD8+ T cells, CD40-Lenhanced CTL generation by PBMC and purified T cells, but not by CD8+ Tcells. Analysis of cytokine secretion using a CTLL assay for IL-2 or anELISA for IFN-γ indicated that CD4+ cells costimulated with CD40-Lproduced 5 to 10-fold more IFN-γ and IL-2 than CD8+ cells. Moreover,CD40-L stimulated CD4+ cells were induced to become cytolytic in alectin-mediated killing assay, whereas IL-2 costimulated both CD8+ andCD4+ cells to become cytolytic.

These data demonstrate that, in addition to accessory moleculesexpressed by antigen presenting cells, membrane proteins may beimportant in T-T cell interactions. The function of CD40-L may be toenhance the expansion of activated T cells within a proliferating T cellclone in a paracrine fashion.

1. A monoclonal antibody that binds to mouse CD40L encoded by vectorpDC406-mCD40-L having ATCC Accession No.
 68872. 2. A pharmaceuticalcomposition comprising a monoclonal antibody according to claim 1, and apharmaceutically acceptable carrier.
 3. A hybridoma which secretes amonoclonal antibody that binds to mouse CD40L encoded by vectorpDC406-mCD40-L having ATCC Accession No. 68872.